CaP coated mesoporous polydopamine nanoparticles with responsive membrane permeation ability for combined photothermal and siRNA therapy

Accepted Manuscript Full length article CaP coated mesoporous polydopamine nanoparticles with responsive membrane permeation ability for combined photothermal and siRNA therapy Zhenqiang Wang, Liucan Wang, Neeraj Prabhakar, Yuxin Xing, Jessica M. Rosenholm, Jixi Zhang, Kaiyong Cai PII: DOI: Reference:

S1742-7061(19)30013-3 https://doi.org/10.1016/j.actbio.2019.01.002 ACTBIO 5858

To appear in:

Acta Biomaterialia

Received Date: Revised Date: Accepted Date:

6 October 2018 11 December 2018 2 January 2019

Please cite this article as: Wang, Z., Wang, L., Prabhakar, N., Xing, Y., Rosenholm, J.M., Zhang, J., Cai, K., CaP coated mesoporous polydopamine nanoparticles with responsive membrane permeation ability for combined photothermal and siRNA therapy, Acta Biomaterialia (2019), doi: https://doi.org/10.1016/j.actbio.2019.01.002

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CaP coated mesoporous polydopamine nanoparticles with responsive membrane permeation ability for combined photothermal and siRNA therapy

Zhenqiang Wang,a Liucan Wang,a Neeraj Prabhakar,b Yuxin Xing,a Jessica M. Rosenholm,b Jixi Zhang, a,* Kaiyong Caia

a Key

Laboratory of Biorheological Science and Technology, Ministry of Education,

College of Bioengineering, Chongqing University, No. 174 Shazheng Road, Chongqing 400044, China. E-mail: [email protected]; Tel: +86 18623316698 b

Pharmaceutical Sciences Laboratory, Faculty of Science and Engineering, Åbo

Akademi University, Tykistökatu 6A, Turku 20520, Finland

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Abstract: Combined photothermal and gene therapy provides a promising modality toward cancer treatment, yet facile integration and controlled codelivery of gene payloads and photothermal conversion agents (PTCAs) remains a great challenge. Inspired by the robust wet adhesion of marine mussels, we present a rationally designed nanosystem constructed by using hybrid mesoporous polydopamine nanoparticles (MPDA) with sub-100 nm sizes and a high photothermal conversion efficiency of 37%. The surface of the particles were modified with tertiary amines by the facile Michael addition/Schiff base reactions of PDA to realize high siRNA loading capacity (10 wt%). Moreover, a successful calcium phosphate (CaP) coating via biomineralization was constructed on the cationic nanoparticle to prohibit premature release of siRNA. The CaP coating underwent biodegradation in weakly-acidic subcellular conditions (lysosomes). The synergistic integration of tertiary amines and catechol moieties on the subsequently exposed surfaces was demonstrated to feature the destabilization/disruption ability toward model cellular membranes via the greatly enhanced interfacial adhesion and interactions. Consequently, sufficient permeability of lysosomal membranes, and in turn, a high lysosomal escape efficiency, was realized, which then resulted in high gene silencing efficiencies via sufficient cytosolic delivery of siRNA. When an efficient knocking down (65%) of survivin (an inhibitor of apoptosis proteins) was combined with a subsequent photothermal ablation, remarkably higher therapeutic efficiencies were observed both in vitro and in vivo, as compared with monotherapy. The system may help to pave a new avenue on the utilization of bio-adhesive surfaces for handling the obstacles of combined photothermal 2

and gene therapy.

Keywords: siRNA delivery, photothermal therapy, porous polydopamine nanocarriers, bioadhesion, lysosomal escape

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1. Introduction Combined cancer therapy, which integrates several therapeutic modalities into a single system with the assistance of nanotechnology, has represented a great potential and experienced solid progress in overcoming the limitation of monotherapy [1]. Among the exploited strategies up to now, the combination of photothermal therapy (PTT) and siRNA based gene therapy (GT) has attracted increasing attention due to the superiority in increasing therapeutic efficacy and decreasing side effects [2-4]. In this system, facile integration and controlled codelivery of two therapeutic agents, i.e. siRNA payloads and photothermal conversion agents (PTCAs), was pivotal to the improvement of the overall therapeutic outcome. To this end, the idea of using porous PTCAs was considered as a significant success in simplifying the integration, as well as realizing large loading and effective protection of siRNA by the pore space [5]. Notwithstanding the first demonstration of utilizing mesoporous carbon nanoparticles [5], there still exist rationaldesign challenges in terms of easier surface functionalization, and efficient escape of the nanocarriers from lysosomes after cell uptake, the latter of which has long been recognized as a major bottleneck in cytosolic delivery of siRNA [6-8]. Recent subversive insights suggested that the classical polyplex-mediated compartmental escape actually relies on the time-dependent membrane permeabilization (via multiple transient pores) by tight apposition of the polyplex and the inner-lysosomal membrane [9, 10]. However, the hydration repulsion between membranes and polar surfaces reduces the mutual perturbation of biomolecular assemblies in the congested cellular environment [11, 12]. Consequently, the abundant body fluids and water in organelles severely hamper the adhesion of siRNA vehicles to inner membrane surfaces via the hydration layers, which has not received enough attention in the past [6, 7, 13]. In this regard, the marine mussel byssal plaque has become a star model for the biomimetic wet adhesion because of the adhesive catecholic amino acid called 3,4dihydroxyphenylalanine (Dopa) which can form various types of interfacial interactions such as hydrogen bonding, metal chelation, π-π and/or cation-π interaction [14, 15]. Mussel-inspired polydopamine (PDA), has attracted immense attention in the community of drug delivery nanocarriers, owing to its advantages in wet adhesion, 4

photothermal conversion, and easy functionalization [16]. Among representative breakthroughs, mesoporous PDA nanoparticles are being developed by our group and others [17, 18], while findings in PDA-assisted biomineralization [19-21] holds a great promise in simple surface encapsulation. Besides, continuous research efforts are also being devoted to exploring new application properties originated from PDA’s bioadhesive feature. Noteworthily, the binding strength of Dopa containing molecules to surfaces was demonstrated to be significantly enhanced by neighboring positively charged lysine or arginine residues [22, 23]. Molecular-level insights into the adhesive mechanisms attributed such a synergy to the role of catechols and amines in cooperative displacing surface-bound salt ions, and in turn the hydration, which allows for effective adhesion and interfacial interactions [24, 25]. Therefore, we anticipated that surface modification of amines on porous PDA-based nanocarriers would address the challenge in lysosomal escape. Herein, we report an efficient siRNA-delivery and PTT system of hybrid mesoporous nanoparticles integrating functions of biomimetic wet-adhesion and biomineralization encapsulation via the combination of the instinct properties from bio-inspired PDA. As shown in Scheme 1, mesoporous PDA nanoparticles (MPDA) with sub-100 nm sizes were prepared as a PTCA with the surface modification of tertiary amines, which facilitated the siRNA loading inside mesopores. Premature release of siRNA was prohibited by calcium phosphate (CaP) coating, which was realized by PDA-induced biomineralization. Facilitated by efficient cellular uptake, pH-responsive CaP degradation, and destabilization of lysosome membrane, an efficient lysosome escape was subsequently demonstrated by in vitro studies. As a consequence, high gene silencing efficiencies can be obtained. The efficiency in siRNA induced therapy was revealed by downregulation of survivin (an inhibitor of apoptosis proteins). The subsequent photothermal ablation resulted in substantially improved therapeutic effectiveness. The novel delivery system is expected to provide new pathways and considerations on the utilization of bio-adhesive surfaces for nanocarriers mediated GT and PTT combination.

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Scheme 1 Schematic illustration on the preparation of hybrid mesoporous nanoparticles (MPDA) integrating functions of PTCA, biomimetic wet-adhesion, and biomineralization encapsulation for combined photothermal and gene therapy. 2. Experimental section 2.1 Materials Unless otherwise noted, all reagent-grade chemicals were used as received, and distilled water was used for the preparation of all aqueous solutions. 2Morpholinoethanesulfonic acid (MES), Ethanol (AR) and acetone (AR) were purchased from Fluka. Pluronic® F-127 and FITC-dextran (M.W. 10 000) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Dopamine hydrochloride (98%, AR), fluorescein isothiocyanate (FITC, 90%), tris (hydroxymethyl) aminomethane (Tris, 99.9%), Acridine Orange， N,N-dimethylethylenediamine (DMEA) and 1,3,5-trimethylbenzene (TMB, AR, 97%) were purchased from Aladdin 6

Industrial Co., Ltd. (Shanghai, China). The hepatocelluar liver carcinoma (Hep-G2) was purchased from Bogoo Biological Technology Co., Ltd (Shanghai, China). SYBR Green I was purchased from Takara Biotechnology Co., Ltd (Dalian, China). Ammonium persulphate (APS), 29 : 1 acrylamide/bisacrylamide 30% gel stock solution, 10× TBE (Tris-borate-EDTA) buffer, crystal violet and N,N,Ntetramethylethylenediamine (TEMED) were purchased from Solarbio Technology Co., Ltd (Beijing, China). SiRNA targeting survivin mRNA (sense sequence: 5’GAAUUUGAGGAAACUGCGA-3’,

antisense

sequence:3’-

CUUAAACUCCUUUGACGCU-5’) [26] and scrambled siRNA (siN.C.) were purchased from Gene Pharma Technology Co., Ltd (Shanghai, China). All Stars Hs Cell Death Control siRNA was purchased from Qiagen (Qiagen GmbH, Germany). Cell counting Kit-8 (CCK-8) was purchased from Dojindo Chemical Inc. (Shanghai, China). LysoTracker Red and calcein-AM/PI double stain kit were purchased from Yeasen Biological (Shanghai, China). Proteinase K, One Step TUNEL Apoptosis Assay Kit and Fluo-3 AM were purchased from Beyotime Institute of Biotechnology (Shanghai, China). Antibodies and secondary antibody were purchased from Cell Signaling Technology Inc. (Danvers, MA, USA). Alanine transaminase (ALT) detection kit, aspartate transaminase (AST) detection kit, serum creatinine (CREA) detection kit and blood urea nitrogen (BUN) were purchased from Jiancheng Bioengineering Institute (Nanjing, China). 2.2 Synthesis of MPDA The MPDA particles were prepared by a one-pot synthesis method reported by our group [17]. In a typical experiment, 0.36 g of F127 and 0.36 g of TMB were firstly dissolved in a mixture of H2O (65 mL) and ethanol (60 mL). After 30 minutes of stirring, a solution of 90 mg of TRIS dissolved in 10 mL of H2O was introduced into the mixture, followed by the addition of 60 mg of dopamine hydrochloride. The reaction mixture was stirred at room temperature for 24 h, and then the product particles were separated by centrifugation. The template removal was performed by solvent extraction. 2.3 Surface modification of MPDA by N,N-dimethylethylenediamine (DMEA) 7

MPDA was modified by N,N-dimethylethylenediamine (DMEA) bearing a tertiary amine group for siRNA loading. Typically, 1 mg of MPDA was suspended in 2 mL of Tris buffer (pH 8.5). Subsequently, different mount of DMEA (0.1-12.8 mg mL-1) was added to the suspension and the mixture was stirred at room temperature for 1 h. Finally, the DMEA modified MPDA was retrieved from the suspension by centrifugation. 2.4 Photothermal performance studies The photothermal performance of DMEA modified MPDA nanoparticles was investigated using an 808 nm laser (Mid-River Company, China) and an E40 IR image system (FLIR, USA). An amount of 1 mL of DMEA modified MPDA nanoparticles with different concentrations (0, 10, 20, 40, 100 μg mL-1) were irradiated with an 808 nm laser (0.5 W cm-2). The temperature was recorded on a digital thermometer with a thermocouple probe (HH806AU, OMEGA, USA). 2.5 SiRNA loading and biomineralization induced CaP coating The loading of siRNA into the mesoporous of DMEA modified MPDA was conducted in 2-Morpholinoethanesulfonic acid buffer (MES buffer, 10 mM, pH 5.0). SiRNA was mixed with 1 mg DMEA modified MPDA in 1 mL MES buffer (pH 5.0) with different w/w ratios (siRNA : MPDA = 0.1 : 1, 0.3 : 1 and 0.5 : 1). Subsequently, the mixture was homogenized by continuous vibration for 40 min and siRNA loaded DMEA modified MPDA (MPDA-siRNA) was separated by centrifugation. For biomineralization induced CaP coating, MPDA-siRNA (1 mg) was suspended in simulated body fluid (SBF) (1.5×, 2 mL) (Na+, 213.0; K+, 7.5; Mg2+, 2.25; Ca2+, 3.75; Cl-, 221.7; HCO3-, 6.3; HPO42-, 1.5; SO42-, 0.75 mM), and the mixture was stirred at 37 °C for 48 h. Finally, the obtained CaP coated MPDA-siRNA (MPDAsiRNA@CaP) nanoparticles were stored at 4 °C before further use. 2.6 Hemolysis assay for studying the membrane destabiliztion Red blood cells (RBCs) were freshly collected in the Hospital of Chongqing University and washed five times with sterile isotonic PBS solution (150 mM, pH=7.4). Then diluted RBC suspension (0.2 mL) was mixed with MPDA or 8

MPDA@CaP suspensions in PBS buffer (0.8 mL, pH 7.4) or MES buffer (0.8 mL, pH 5.0) at various concentrations. After a shorter (3 h) or longer (12 h) incubation time, the samples were then centrifuged at 1800 rpm for 4 min to pellet any remaining intact RBCs. The absorbance of the supernatant from each sample was measured at 545 nm. Hemolysis levels were normalized to both negative and positive controls. In controls, RBCs were incubated in PBS or MES (negative control) and in distilled water (positive control). The percent hemolysis of RBCs was calculated using the following formula: percent hemolysis = ((sample absorbance - negative control absorbance) × 100 / (positive control absorbance - negative control absorbance)). 2.7 Permeability assay of lysosomal membrane after particle uptake Permeability of lysosome membranes was evaluated using the acridine orange (AO) uptake method. As a metachromatic fluorophore, AO becomes protonated within acidic compartments. Lysosomes loaded with AO emit an intense red fluorescence, whereas nuclei and cytosol showed weak diffuse green fluorescence [27, 28]. Hep-G2 cells were seeded onto 6-well plates at density of 1 × 105 per well and incubated at 37 °C for 24 h. And then, cells were incubated with MPDA@CaP (40 μg mL-1) for different time periods. Cultures were incubated with 500 nM AO for 15 min before being sequentially imaged using a confocal laser scanning microscopy (CLSM, LSM 510 META, Olympus). The mean fluorescence intensity (MFI) was detected by flow cytometry (Accuri C6, BD ). 2.8 Co-localization study for the quantitative analysis of lysosome escape To make it clear whether the particles can escape from the lysosomes, Hep-G2 cells were incubated with MPDA-siN.C.@CaP (40 μg mL-1) or FITC-dextran (250 μg mL-1) for 24 h. Here the siN.C. (negative control siRNA) was labeled by SYBR Green I. Then the treated cells were stained with a LysoTracker Red solution for 30 min and imaged with CLSM. For statistical analysis of co-localization, the PSC co-localization plug-in for ImageJ was used to calculate the linear Pearson correlation coefficient (PSC) of red and green fluorescent signals [29-31]. Values of PSC were between -1 (negative correlation) and +1 (positive correlation). 9

2.9 Evaluation on the down-regulation of survivin gene expression Hep-G2 cells were seeded into 6-well plates and cultured at 37 °C for 24 h. The cells were incubated with: (1) the naked siRNA (targeting survivin mRNA); (2) MPDA@CaP (3) MPDA-siN.C.@CaP; (4) MPDA-siRNA@CaP for 48 h. The cellular levels of survivin protein were assessed by using Western Blot. The cell apoptosis was determined using the Annexin V-FITC/PI apoptosis detection. 2.10 In vitro therapeutic efficiency studies The Hep-G2 cells were cultured in a 24-well plate and cultured at 37 °C for 24 h. The cells varied under different conditions: (1) only saline treament; (2) only NIR treatment for 10 min; (3) incubated with MPDA-siRNA@CaP for 48 h; (4) incubated with MPDA@CaP for 48 h and irradiated with an 808 nm laser at 0.5 W cm-2 for 10 min; (5) incubated with MPDA-siRNA@CaP for 48 h and irradiated with an 808 nm laser for 10 min. Finally, the live/dead cell staining assay was conducted with calceinAM/PI double stain kit, and the cells viability was measured using the CCK-8 assay. 2.11 In vivo study The animal experiments were strictly conducted according to the regulations of the Institutional Animal Care and Use Committee of China with the approval of the ethics committee of Third Military Medical University (SYXK-PLA-20120031). Male nude mice of about 4-6 weeks old were bought from the Animal Laboratory of Daping Hospital (Chongqing). For the establishment of tumor xenograft models, the nude mice were subcutaneously injected with 2 × 107 Hep-G2 suspended in 100 µL of PBS. When the tumor size reached around 80 mm3, all tumor-bearing mice were equally subjected to 7 groups (n = 5): (1) mice received an intratumoral injection of 100 µL physiological saline solution ( NaCl, 0.9 wt%) as the control group; (2) mice received a laser irradiation (10 min at 0.5 W cm-1) every other day; (3) mice received an intratumoral injection of MPDA@CaP at a dose of 2 mg kg-1 every other day; (4) mice received an intratumoral injection of MPDA-siN.C.@CaP at a dose of 2 mg kg-1 every other day; (5) mice received an intratumoral injection of MPDA-siRNA@CaP at a dose of 2 mg kg-1 every other day; (6) mice received an intratumoral injection of 10

MPDA@CaP at a dose of 2 mg kg-1 every other day, followed by 10 min of laser irradiation after 24 h of injection; (7) mice received an intratumoral injection of MPDA-siRNA@CaP at a dose of 2 mg kg-1 every other day, followed by 10 min of laser irradiation after 24 h of injection. The tumor sizes and body weights of nude mice were measured just before each administration. After 5 times treatments, the mice were sacrificed. 2.12. Statistical analysis Data was manifested as mean ± standard deviation (SD) of 3 independent trails. Statistical analysis was performed by Student t-test for two groups, and one-way ANOVA for multiple groups. A P value lower than 0.05 was considered statistically significant, which was represented in the following figures as *p < 0.05, **p < 0.01 and ***p < 0.001. 3 Result and Discussion 3.1 Preparation, characterization, and siRNA loading of MPDA nanocarriers MPDA was first synthesized based on the assembly of primary PDA particles and Pluronic F127 stabilized emulsion droplets on water/1,3,5-trimethylbenzene (TMB) interfaces, according to the procedure in a previous study by us [17]. To impart the particle surface with positive charges and high affinity of siRNA within mesopores, MPDA was modified by N,N-dimethylethylenediamine (DMEA) bearing two different amine groups. Herein, the primary amine of DMEA facilitates the conjugation towards PDA by the Michael addition/Schiff base reactions [16, 32], while tertiary amines provide positive charges for electrostatically binding siRNA, protecting nucleic acids from enzymatic degradation, and also for interacting with lysosomal membranes inside cells. Typical transmission electron microscopy image (TEM, Fig. 1a) and scanning electron microscopy images (SEM, Fig. S1a and b) reveal that the DMEA modified MPDA sample consists of spherical particles with an average diameter of ∼90 nm. A close examination of the surface topography suggests that there are irregular pores with diameters below 10 nm. As shown in Fig. 1b, the nitrogen adsorption–desorption 11

plot exhibits a type-IV isotherm of mesoporous materials. The specific surface area was determined to be 45 m2 g-1 for DMEA modified MPDA. A wide capillary condensation step with a type-H4 hysteresis loop in the P/P0 range of 0.4–0.8, corresponding to a relatively broad pore size distribution in the range of 3–30 nm and a peak pore size of 5 nm (Fig. 1c). A substantial increase in zeta potential of DMEA modified MPDA from -30 mV to 3.2 mV (Fig. 1d) was observed by increasing the employed DMEA concentration from 0 mg mL-1 to 2 mg mL-1 in the modification solution (Tris buffer, pH 8.5). Zeta potentials of MPDA increased slowly afterwards and reached 10.8 mV (at 12.8 mg mL-1 of DMEA), implying that the DMEA density on MPDA was saturated. Fluorescamine quantification of DMEA indicated that the amount of DMEA present on MPDA nanoparticles is ~2.25 mmol g-1.

Fig. 1. Characterization of MPDA particles: (a) TEM image; (b) Nitrogen sorption isotherm; (c) Corresponding pore size distribution deduced from the desorption branch of the isotherm; (d) Effect of different concentration of DMEA on zeta 12

potential of MPDA; (e) Temperature increasing curves of DMEA modified MPDA nanoparticles-dispersed aqueous suspension at varied concentrations (100, 40, 20, 10 and 0 μg mL-1) under irradiation using an 808 nm laser (0.5 W cm-2). (f) Recycling heating profile of DMEA modified MPDA nanoparticles suspension dispersed in water (40 μg mL-1) with an 808 nm laser irradiation (0.5 W cm-2) for three laser on/off cycles. The photothermal conversion performance of the DMEA modified MPDA nanoparticles induced by 808 nm laser irradiation (0.5 W cm-2) was measured. The temperature of a 1 mL solution containing 100 μg mL-1 of particles was increased by 10 °C in 780 s (Fig. 1e). The particles showed concentration-dependent temperature elevation between 2.4 °C and 10 °C at a particle concentration range of 10 to 100 μg mL-1, while the temperature of deionized water was increased by only 1.3 °C. The photothermal performance of the particles does not show any significant deterioration during three laser on/off cycles (Fig. 1f), indicating the high stability of the particles as a durable PTCA for PTT cancer treatment. By UV-vis absorption spectra (Fig. S2a), the normalized adsorption intensity over the lengh of the cell (A/L) at varied concentrations (C) was determined, and the extinction coefficent at 808 nm was measured to be 10.14 L g-1 cm-1 (Fig. S2b). Furthermore, the photothermal conversion efficiency (η) of DMEA modified MPDA was calculated to be 37% based on the results of time constant for heat transfer and the maximum steady-state temperature (Fig.S2c and d). To investigate the loading capability of DMEA modified MPDA as siRNA nanocarriers, a retardation assay by agarose gel electrophoresis was performed after mixing MPDA with varying amounts of scrambled siRNA (w/w ratio, MPDA/siRNA=1 : 0.1, 1 : 0.3, 1 : 0.5) in MES buffer (pH 5.0), and the results are shown in Fig. S3a. Complete retardation of siRNA was observed at a w/w ratio of 1:0.1, suggesting the positively charged MPDA could load siRNA completely at this ratio. The absence of UV-vis absorbance in the supernatant of binding buffer (Fig. S3b) also confirmed this observation. The N/P ratio of DMEA modified MPDA to siRNA is 7.1, which is comparable to the results in recent reports employing 13

mesoporous particles as siRNA nanocarriers [33, 34]. 3.2 Construction of CaP coated particles by biomineralization To prevent uncontrolled and premature release of siRNA from the mesopores, MPDA-siRNA nanoparticles were covered with pH controlled, absorbable calcium phosphate (CaP) nanocoatings as pore blockers which would facilitate the release of entrapped siRNAs within acidifying intracellular compartments such as lysosomes. It has been reported that polydopamine on the surface can play an important role in the biomineralization, where the abundant catechol moiety strongly binds metal ions and leads to hydroxyapatite formation by co-precipitation of calcium and phosphate ions [35]. MPDA particles were incubated under stirring in simulated body fluid (SBF) at 37 °C for 48 h. SEM image (Fig. 2a) shows the surface morphological change of MPDA after mineralization. MPDA@CaP sample possesses the same spherical morphology, but a continuous external surface, as well as an increased average diameter of ∼100 nm. A closer look at the inner structures of the particles at high magnification (Fig. 2b) shows that the open mesopores disappeared, indicating mesopore sealing during the biomineralization process. SEM-associated energydispersive X-ray spectroscopy (EDS) analysis showed that mineralized surfaces predominantly contained Ca and P, which should be originated from CaP minerals. The Ca/P molar ratio was estimated to be about 1.7, which is close to the theoretical ratio of hydroxyapatite (Ca/P=1.67). The X-ray photoelectron spectroscopy (XPS) analysis (Fig. 2c) also demonstrated that nanoparticles had characteristic peaks of Ca (2p) and P (2p) elements. Wide-angle X-ray diffraction (XRD) patterns (Fig. 2d) show that MPDA@CaP exhibited characteristic sharp peaks of hydroxyapatite, i.e. (211) and (002). Additionally, strongly negative zeta potential (-30 mV) and larger hydrodynamic diameter (~220 nm) of the coated particle (Fig. S4) were observed. All the evidence confirmed that the surface mineralization process induced the nucleation and growth of hydroxyapatite nanocoatings on mesoporous PDA surfaces [35, 36].

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Fig. 2. Characterization of MPDA@CaP particles: (a) SEM image and SEMassociated EDS spectra; (b) TEM image; (c) XPS analysis; (d) Wide-angle XRD patterns; (e) Biomineralization kinetic profiles by monitoring residual Ca2+ in supernatants of SBF; (f) Evaluation of premature release of siRNA in the process of biomineralization by measuring the fluorescence of scrambled siRNA in the supernatant. We further investigated the influence of siRNA loading on the CaP coating of the particles. TEM characterization showed that there was no discernable change on the particle morphology. However, the kinetic profiles of the biomineralization (Fig. 2e), as evaluated by monitoring the redidual Ca2+ in SBF via the Arsenazo III method (Fig. S5) [37], show that the equilibrium time requied for MPDA and MPDA-siRNA was 11 h and 5 h, respectively. The result indicates an accelerated formation of CaP coating in the presence of siRNA, which should be explained by chemical similarity 15

between the phosphate backbone of the oligonucleotide and the calcium phosphate [38-40], as well as the electrostatic interactions between oligonucleotides and Ca2+. As shown in Fig. 2f, no fluorescence was observed in the supernatant of the coating suspension, revealing that there was no siRNA release in the process of biomineralization. To verify the possible protection effect of the siRNA by the mesopores of MPDA and CaP coating, polyacrylamide gel electrophoresis analysis was performed. As shown in Fig. 3a, free siRNA was cleaved to the bands of short lengths (lane 2) when incubated with RNase. In contrast, no siRNA release/degradation was found when MPDA-siRNA@CaP treated with heparin (lane 3) or RNase (lane 4), which is most likely related to the blockage effect of CaP coating. To mimic the lysosomal environment, MPDA-siRNA@CaP was incubated with MES buffer (pH 5.0) for 24 h. When the obtained suspension was treated with heparin, siRNA release was observed (lane 5), suggesting the disintegration of the CaP coating under weakly acidic conditions. In comparison, no release was found after treatment in the buffer with a pH of the physiological environment (PBS pH 7.4, lane 6). Furthermore, we evaluated the protection performance of DMEA modified MPDA, and the results are shown in Fig. S6. Without the protection of CaP coating, siRNA release/degradation was found when MPDA-siRNA treated with heparin (lane3) or RNase (lane 4). Besides, Sustained release was found after treatment in the PBS buffer (pH 7.4) for 0.5 h (lane 5), 1 h (lane 6), 2 h (lane 7) and 4 h (lane 8). These results further demonstrated the importance of CaP coating.

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Fig. 3. Protection effect of siRNA and responsive degradation of CaP. (a) Polyacrylamide electrophoresis analysis for the protective effect of siRNA by MPDA@CaP: lane 1: FAM-siRNA; lane 2: FAM-siRNA after RNase I digestion; lane 3: MPDA-siRNA@CaP treated with heparin; lane 4: MPDA-siRNA@CaP after RNase I digestion; lane 5: MPDA-siRNA@CaP treated with MES (pH 5.0) for 24 h and then incubated by heparin; lane 6: MPDA-siRNA@CaP treated with PBS (pH 7.4) for 24 h and then incubated by heparin. (b) TEM image of MPDA@CaP particles after treated with MES (pH 5.0) for 24 h. (c) Kinetics of calcium dissolution from MPDA-siRNA@CaP under pH control (**p < 0.01). (d) Zeta potentials of MPDA@CaP particles after treated with MES (pH 5.0) for different time periods. Dissolution of CaP coating from MPDA@CaP was then investigated, regarding the pH-dependent stability of CaP [41]. As shown in the TEM image (Fig. 3b), the CaP coating disappeared after treated with MES buffer (pH 5.0), accompanying with the reemergence of the porous structure of MPDA. Additionally, the pH-dependent dissolution kinetics of CaP coating from MPDA-siRNA@CaP were also investigated. As shown in Fig. 3c, the mineral coating underwent minimal dissolution (< 5%) under the physiological pH condition (PBS, pH 7.4). In contrast, a gradual increase of the released Ca2+ up to 47% in 12 h was observed under the low pH condition (MES, pH 17

5.0), implying accelerated dissolution of CaP coating. The pH-induced progressive dissolution was also supported by the variation in the zeta potentials of MPDA@CaP during incubation (Fig. 3d). The negative surface charge resulting from CaP was changed to be neutral after 24 h of incubation. The gradually exposed surfaces of DMEA modified MPDA with positive charges resulted in the reverse of the zeta potential of MPDA@CaP at 48 h of incubation. Hence, it was expected that the CaP coating can prevent premature siRNA release at extracellular conditions, and degrade under subcellular conditions (lysosomes) to expose MPDA-siRNA. 3.3 Cytotoxity and cellular uptake The potential nonspecific toxicity of carrier is always a great concern for nanomaterials used in biomedicine. Biocompatibility of siRNA loaded MPDA with or without CaP coating particles was determined by incubating with Hep-G2 for 24 h. As revealed by the CCK-8 assay (Fig. S7a and b), the cell viability of Hep-G2 remained above 80% and 90%, when they were treated with two kinds of particles with concentrations up to 100 μg mL-1. The endocytosis pathway of MPDA@CaP was then determined by monitoring the change in the fluorescence intensity of siN.C. (labeled with SYBR Green I, loaded in MPDA@CaP) after incubating the cells in presence of different uptake inhibitors for specific pathways. Cells were separately pretreated with chlorpromazine (CPZ, 10 μg mL-1), nystatin (20 μg mL-1) and ethylisopropyl amiloride (EIPA, 133 μg mL-1) for 1 h before siRNA transfection. In general, CPZ and nystatin were used for the inhibition of clathrin-mediated endocytosis and caveolae-mediated endocytosis, and EIPA was used to inhibit the macropinocytosis. As shown in Fig. S8, EPIA did not affect the cellular uptake of MPDA-siN.C.@CaP, suggesting that MPDAsiN.C.@CaP was not taken up by means of macropinocytosis. However, the cellular uptake of MPDA-siN.C.@CaP was inhibited to a certain extent when CPZ or nystatin was used alone. These results demonstrated that MPDA-siN.C.@CaP was mainly internalized by the patterns of both clathrin-mediated endocytosis and caveolaemediated endocytosis, but not macropinocytosis. As reported very recently [42], in 18

most cell types caveolar endocytosis leads cargo to the lysosomal compartment if they were not recycled back to the cell surface by early lysosomes. As a consequence, MPDA@CaP nanocarriers would then meet the great challenge of escape from the congested lysosomal environment filling with abundant body fluids and water. 3.4 Particle-membrane interaction and lysosomal escape via bioadhesion In order to demonstrate the potential of the bio-adhesion design in our nanocarriers for lysosomal escape, the interactions of the particles with artificial lysosomal membranes were then investigated. Red blood cells (RBCs) have been frequently used as a classic model an artificial lysosomal membrane with a biological relevance, owing to the existence of proteins and lipid asymmetry inside their cell membrane [9]. In order to investigate the membrane-penetrating ability of the nanocarriers in a quantitative way, hemolysis assay was carried out. As shown in Fig. 4a, the hemolysis percentages of RBCs incubated with DMEA modified MPDA in different buffer condition (PBS, pH 7.4 and MES, pH 5.0) display very different manner. No apparent hemolysis was detected after 3 h of incubation at particle concentrations up to 100 μg mL-1 in PBS buffer and only 14% hemolysis was observed at 200 μg mL-1. However, in MES buffer, a substantial increase in the hemolysis was observed with the increasing particle concentration. Strikingly, over 60% of RBCs were lysed by the particles at 200 μg mL-1. As reported, water disrupts interactions on polar surfaces by forming hydration layers that impede intimate contacts between materials and surfaces [11, 12], and catechols alone in PDA are insufficient to break the hydrated salt layer [22, 43]. In our nanocarriers system, it is highly possible that protonated tertiary amines caused by low pH buffer, and catechols on the external surface of DMEA modified MPDA, cooperatively interacted with RBC membrane after efficient wet adhesion and consequently resulted in hemolysis [22]. Subsequently, persistent membrane destabilization can lead to the transient pore formation in the membrane, resulting in the leakage of hemoglobin [9, 44].

19

Fig. 4. Hemolysis percentage of RBCs after incubated with MPDA or MPDA@CaP suspensions (ranging from 20 to 200 μg mL-1) in different buffers (PBS pH 7.4, MES pH 5.0) for different times (insets: photographs of RBCs after treatments): (a) RBCs incubated with DMEA modified MPDA for 3 h; (b) RBCs incubated with MPDA@CaP for 3 h; (c) RBCs incubated with MPDA@CaP for 12 h (**p < 0.01, ***p < 0.001). (d) Schematic illustration for the process of hemolysis induced by MPDA@CaP. Evaluation of lysosome disruption capability of MPDA@CaP: (e) Negative control without MPDA@CaP treatment and acridine orange (AO) staining; (f) Positive control without MPDA@CaP treatment but with AO staining; Fluorescence microscopy images of cells incubated with MPDA@CaP (40 μg mL-1) for (g) 6 h, (h) 12 h, (i) 24 h and stained with AO. The scale bar in (e-i) represents 25 μm. Fig. 4b and c show the hemolysis percentages of RBCs caused by MPDA@CaP after different incubation times (3 h versus 12 h). As compared with MPDA, MPDA@CaP did not induce obvious hemolysis after the same short incubation period of 3 h (Fig. 4b), no matter what kind of buffer was used. The hemolysis enhanced after 12 h of incubation, only 17% hemolysis was observed for concentrations up to 200 μg mL-1 in PBS buffer. Surprisingly, 41% hemolysis was reached in MES buffer 20

(Fig. 4c). On the basis of the above results, we conformed that the dissolution of CaP coating at acidic environments can lead to the exposure of DMEA modified MPDA surface, and in turn, its favorable interactions with biomembranes (Fig. 4d). To obtain an indirect measurement of the extent of particle adsorption, an optical technique of fluorescence resonance energy transfer (FRET) was utilized. As shown in Fig. S9, the mixture of DiI labeled liposomes and FITC-labeled MPDA showed an apparent FRET emission of DiI at 565 nm after the FITC excitation at 420 nm, suggesting the particle binding on the surface of liposomes by bioadhesion. Significantly, a stronger FRET was determined in the case of DMEA modified MPDA particles, which should be related to the syngersitic effect of tertiary amines on facilitating the adhesive properties of catechols. Encouraged by the promising results on the interaction between MPDA@CaP and the artificial lysosomal membranes, we then carried out the stability assay of lysosomal membrane in cancer cells, in order to evaluate lysosomal escape ability. Acridine orange (AO) was utilized as a marker to trace the integrity of the lysosomes [27, 45, 46]. Hep-G2 cells without AO staining were used as negative control (Fig. 4e), while cells with AO staining but without MPDA@CaP treatment were used as a positive control (Fig. 4f). Herein, acidic organelles (lysosomes) of cells could be stained to red by AO. After treated with MPDA@CaP (Fig. 4g, h and i), the red spots decreased, and almost disappeared over time, implying the increase in the permeability of the lysosome membranes. The fluorescence intensity of red acidic organelles was also quantified by flow cytometry in FL-3 channel. As shown in Fig. S10, cells treated with MPDA@CaP for 6 h show a significant increase (50.55%) in the number of pale cells, as compared to positive control (without MPDA@CaP treatment, AO stained, 100%). A large increase in the number of pale cells was observed in cells treated with MPDA@CaP for 12 h (79.90%) compared to 6 h. Furthermore, there was only a slight increase in the number of pale cells after 24 h (86.13%) compared to 12 h, which indicated that the membrane permeation was increased with time. As shown in Fig. S11, the intracellular Ca2+ concentration was increased after the cells were treated with MPDA@CaP. These results indicate that 21

CaP coating was dissolved at acidic organelles, which may lead to the pore formation in the lysosomal membrane by the exposed surfaces of DMEA modified MPDA. Having demonstrated the high ability of membrane destabilization, we examined the lysosomal escape efficiency of MPDA@CaP by a co-localization study using fluorescence-labeled siN.C., along with high resolution microscopy images. As shown in Fig. 5a, both punctate and diffused green fluorescence patterns at the SYBR Green I (siN.C.) channel were found inside cells after 24 h of incubation. There exists a certain degree of co-localization of SYBR Green I with lysosomes when Lysotracker Red was used to stain the lysosomes of cells. To quantify the co-localization results, we calculated the linear Pearson correlation coefficient (PSC) for the pixels representing the fluorescence signals in both channels. FITC-dextran was used as a positive control for endocytic uptake without lysosomal escape (Fig. 5b) [47]. The fluorescence value of pixels across the two channels were additionally depicted in an intensity scatterplot (Fig. 5c and d). In MPDA-siN.C.@CaP treated cells, the siN.C. signals and Lysotracker Red signals were mostly separate with a lower PSC value of 0.21, compared with FITC-dextran trapped inside lysosomes (with a PSC of 0.9, Fig. 5e). These results are in line with the membrane permeability study, further supporting a high degree of lysosomal escape for MPDA@CaP particles through membrane destabilization via strong bio-adhesion. Another possible factor which might contribute to the efficient escape is the proton sponge mechanism generated by the tertiary amines. However, recent studies have proved that this type of escape relies on protonation-induced membrane permeabilization by tight apposition of the positively charged species and the inner-lysosomal membrane.[10] This further supports the significant role of bioadhesive binding on the lysosomal escape of MPDA by the synergistic integration of tertiary amines and catechol moieties.

22

Fig. 5. Confocal laser scanning microscopy (CLSM) images of Hep-G2 cells incubated with (a) 40 μg mL-1 of siN.C. (labeled with SYBR Green I) loaded MPDA@CaP or (b) 250 μg mL-1 of FITC-dextran for 48 h. (c) Intensity scatterplot of fluorescence signals from (a), representing SYBR Green I (green) and LysoTracker red (red). (d) Intensity scatter plot of fluorescence signals from (b), representing FITC-dextran (green) and LysoTracker red (red). (e) Bar chart to illustrate the PSC coefficients from (c, d) (***p < 0.001). The scale bar in (a) and (b) represents 25 μm. 3.5 Gene silencing mediated by MPDA-siRNA@CaP In light of the efficient lysosomal escape, we continued to investigate the silencing effects of the model target genes by both a fluorescence method and a nonfluorescence method. First, siRNA (targeting EGFP mRNA) transferred by Lipo2000 and MPDA@CaP could significantly knockdown EGFP gene expression (Fig. S12a, b and c). Meanwhile, the fluorescence intensity of EGFP was quantified by flow cytometry. As shown in Fig. S12d, lower percentage of EGFP expressing cells (10%) were left when the cells were treated with MPDA-siRNA@CaP, compared to 28% (treated with Lipo2000-siRNA) and 68% (non-treated). Second, we used a 23

commercially available cell-killing siRNA (All Stars Hs Cell Death Control siRNA) to get a more intuitive data. The results were analyzed by the crystal violet staining assay (Fig. S12e) and CCK-8 assay (Fig. S12f). Only 23% area percent of cells were alive after treated with MPDA-siRNA@CaP loaded with All Stars Hs Cell Death Control siRNA for 48 h. The same trend was also indicated by the CCK-8 assay. The great success in silencing model genes encouraged us to apply the nanocarriers for real gene therapy. A therapeutic siRNA targeting survivin mRNA [26] was encapsulated in MPDA@CaP. Survivin is an inhibitor of apoptosis proteins, which functions to negatively regulate cell apoptosis, and is highly expressed in many cancer cells represented by human hepatoma cells and associated with therapy resistance [48]. The level of the protein expression was determined by the Western Blot analysis. As shown in Fig. 6a, compared with other control groups, the MPDAsiRNA@CaP remarkably downregulated the expression of survivin protein to a remarkably low level (~35%). Next, we estimated the apoptosis-inducing effect using the Annexin V-FITC/PI apoptosis detection (Fig. 6b). As expected, the cells after treatment with MPDA-siRNA@CaP showed the strongest apoptosis. The total apoptotic ratio, a sum of early and late apoptotic ratios, was 18.91%, significantly higher than that of other control groups. It is thus confirmed that MPDA@CaP with the high gene-silencing efficiency exhibited a superior apoptosis-inducing capacity.

Fig. 6. In vitro gene silencing efficacy of nanoparticles. (a) Survivin protein expression determined by the Western Blot analysis. (b) Cell apoptosis after treatment with survivin-targeting siRNA loaded MPDA@CaP determined by the Annexin V24

FITC/PI assay. The viable, early apoptotic and late apoptotic cell populations (%) are shown in the lower left, lower right, and upper right quadrants, respectively (**p < 0.01). 3.6 Combined PTT/GT therapy Inspired by the result of gene silence effects, we continued to examine the PTT and GT combination effect of the MPDA-siRNA@CaP in vitro. After the cells were treated with MPDA@CaP and NIR laser irradiation (808 nm, 0.5 W cm-2, 10 min), the lived and dead cells were differentiated by calcein-AM (green) and propidium iodide (PI) (red) co-staining. The control groups including the cells with only saline treatment (Fig. 7a), only NIR laser irradiation (Fig. 7b), and only MPDAsiRNA@CaP treatment (Fig. 7c) were not significantly affected by the treatments. In contrast, most cells were killed by the photothermal ablation by MPDA@CaP (Fig. 7d), owing to the excellent PTT performance of the nanoparticles inducing a temperature elevation of ~9 °C (Fig. 7f). Furthermore, once siRNA and NIR were both applied by the nanocarriers, the amount of dead cells was significantly increased (Fig. 7e). The combined therapy can also induce the lowest cell viability of 5% (Fig. 7g and Fig. S13a), as compared with GT (~85%) and PTT (~45%). Importantly, the combination index (Fig. S13b) analysis at different dosages suggested the apparent synergistic effect for MPDA@CaP based PTT and gene therapy. More importantly, the amount of dead Hep-G2 cells was dependent on the particle-incubation time before the application of NIR irradiation (Fig. S14). More tumor cells were killed by NIR irradiation with a longer MPDA-siRNA@CaP incubation time (from 12 h to 24 h and 48 h), which can be ascribed to the time-consuming process of lysosomal escape and gene silencing. These results clearly demonstrate the remarkable combinedtherapy effects of MPDA-siRNA@CaP in vitro.

25

Fig. 7. Fluorescence live/dead cell images of Hep-G2 cells with different treatments: (a) Saline; (b) NIR; (c) MPDA-siRNA@CaP; (d) MPDA@CaP + NIR; (d) MPDAsiRNA@CaP + NIR. (f) IR thermal images of MPDA-siRNA@CaP treated cells before and after NIR treatments. (g) Viability of Hep-G2 cells with different treatments. Cells were treated for 48 h, and then NIR irradiation (0.5 W cm-2, 10 min) was applied (**p < 0.01, ***p < 0.001). The scale bar in (a-e) represents 100 μm. We used the Hep-G2 tumor-bearing nude mice to explore the therapeutic potential of MPDA-siRNA@CaP in vivo. The body weight of the mice did not show obvious change during the treatment with MPDA@CaP (Fig. S15). As shown in Fig. 8a and Fig. S16, the tumor sizes of the mice receiving an intratumoral injection of saline, NIR, MPDA@CaP or MPDA-siN.C.@CaP, were 5-8 folds larger than the initial sizes after 5 times treatments. For the mono-GT group, the MPDAsiRNA@CaP exhibited stronger effects on inhibiting the tumor growth (3.4 fold larger than the initial sizes). For the mono-PTT group, the tumor was reduced slightly after 10 days, owing to the strong PTT efficiency of MPDA@CaP which elevated the tumor temperature to ~46 °C after 10 min of NIR irradiation (Fig. 8b). Furthermore, 26

the enhanced therapeutic efficiency was observed in the PTT-GT combined treatment group when compared with the control group (p < 0.001), the mono-GT group (p < 0.01), and the mono-PTT group (p < 0.05), where the tumor sizes reduced during two days after treatment. In addition, the histological analysis (Fig. 8c and Fig. S17) confirmed the greater antitumor efficacy but low toxicity for normal organs (heart, liver, spleen, lung and kidney) of MPDA-siRNA@CaP via the combined therapy. Furthermore, the blood biochemistry assay showed that the content of specific indicators of liver and kidney functions, including alanine transaminase (ALT), aspartate transaminase (AST), serum creatinine (CREA) and blood urea nitrogen (BUN), fell within normal ranges (Fig. S18a-d). These were no significant differences between control group and PTT + GT group. These results further demonstrated the biocompatibility of MPDA-siRNA@CaP with no acute toxicity in vivo and supported its potential application in tumor treatment.

Fig. 8. In vivo antitumor efficacy of nanoparticles in Hep-G2 tumor-bearing nude mice. (a) Change in tumor sizes of the tumor-bearing mice during the 10-day 27

treatment

with

saline,

NIR,

MPDA@CaP,

MPDA-siN.C.@CaP,

MPDA-

siRNA@CaP, MPDA@CaP + NIR and MPDA-siRNA@CaP + NIR (*p < 0.05, **p < 0.01, ***p < 0.001). (b) The corresponding temperature elevations at the tumor sites in groups of NIR and MPDA@CaP + NIR laser during laser irradiation. Insets: IR thermal images at the tumor site of Hep-G2-tumor-bearing mice. (c) Histological images of the HE-stained tumor tissues harvested from different treated groups of mice. Scale bar: 100 μm. Conclusion In summary, we have demostrated that the synergistic integration of tertiary amines and catechol groups on surfaces of porous nanocarriers can offer a simple and highly efficient solution to handle the obstacle of combined photothermal and gene therapy on the basis of porous photothermal conversion agents. Application of this nanosystem gave an example on cooperative integration of siRNA delivery and heat generation by a porous photothermal conversion agent (PTCA). Most importantly, the efficient particle-membrane interactions were facilitated by the synergy of surface amines/catechols, and were verified by elaborative characterizations including high hemolysis percentages, increased permeability of lysosomal membranes, and low colocalizaiton of the cargo siRNA and lysosomes. As guaranteed by the critical factors above, the high gene silencing efficacy of siRNA loaded MPDA@CaP was demonstrated by intracellular delivery of EGFP-siRNA, cell-killing siRNA, as well as in vitro/vivo delivery of the therapeutic survivin siRNA, respectively. The subsequently applied photothermal ablation can thus be obtained, which led to high combined-therapy efficiency. This strategy smartly combines porous PTCA with the bioadhesive surface and biomineralization coating, and has great potential for combined therapy in a simple and easily controlled manner.

Acknowledgements This work was supported in part by the National Natural Science Foundation of China (NSFC, Grant No. 51773022, 51502027, 21734002), Basic Advanced Research 28

Project of Chongqing (Grant No. cstc2015jcyjA10051), Graduate research and innovation foundation of Chongqing, China (Grant No. CYB18028), 100 Talents Program of Chongqing University (J. Z.), and Innovation Team in University of Chongqing Municipal Government (CXTDX201601002). Declarations of interest None. Appendix A. Supplementary data Supplementary data to this article can be found online.

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Survivin Treatments for Cancer, Curr. Med. Chem. 17 (2010) 1509-1515. Statement of Significance The highlights of this paper are as follows: 1.

Polydopamine (PDA) based porous photothermal-conversion agent (PTCA) with sufficiently high conversion efficiency was employed to deliver photothermal/gene therapy modalities towards cancer treatment.

2.

CaP coating via PDA-induced biomineralization was constructed to prohibit premature release of siRNA loaded in the pore space of the nanocarriers.

3.

Responsive degradation of CaP also led to the exposure of membrane-lytic surfaces built through the synergistic integration of tertiary amines and catechol moieties, and in turn the significantly enhanced lysosomal escape and cytosol siRNA delivery.

4.

Therapeutic targeting of survivin was successfully applied for activation of apoptosis and programmed cell death. Combined photothermal and gene therapy improved therapeutic effectiveness.

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