tripolyphosphate nanoparticles and their pMDI formulation for survivin siRNA pulmonary delivery

Accepted Manuscript Title: GABAB Receptor Ligand-directed Trimethyl Chitosan/Tripolyphosphate Nanoparticles and Their pMDI Formulation for Survivin siRNA Pulmonary Delivery Authors: Suhui Ni, Yun Liu, Yue Tang, Jing Chen, Shuhan Li, Ji Pu, Lidong Han PII: DOI: Reference:

S0144-8617(17)31108-6 https://doi.org/10.1016/j.carbpol.2017.09.075 CARP 12820

To appear in: Received date: Revised date: Accepted date:

21-6-2017 8-9-2017 23-9-2017

Please cite this article as: Ni, Suhui., Liu, Yun., Tang, Yue., Chen, Jing., Li, Shuhan., Pu, Ji., & Han, Lidong., GABAB Receptor Liganddirected Trimethyl Chitosan/Tripolyphosphate Nanoparticles and Their pMDI Formulation for Survivin siRNA Pulmonary Delivery.Carbohydrate Polymers https://doi.org/10.1016/j.carbpol.2017.09.075 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

GABAB Receptor Ligand-directed Trimethyl Chitosan/Tripolyphosphate Nanoparticles and Their pMDI Formulation for Survivin siRNA Pulmonary Delivery

Suhui Ni1, Yun Liu1, Yue Tang*, Jing chen, Shuhan Li, Ji Pu, Lidong Han.

These authors contributed equally to this work and should be regarded as co-first authors.1 Department of Pharmacy, China Pharmaceutical University, Nanjing 211198, PR China Corresponding author: Yue Tang, Tel: +8613851713608. Fax: +862583271355. E-mail address: [email protected].

Highlights 1. Baclofen groups were integrated into TMC via amide bond formation. 2. Baclofen functionalized nanoparticles increased the uptake of siRNA through the interaction with GABAB receptor resulting efficient gene silencing. 3. Mannitol microparticles was utilized for siRNA pulmonary delivery via HFA-134 based pressurized metered dose inhalers (pMDI).

Abstract The effect of gene silencing by survivin siRNA (siSurvivin) on the proliferation and apoptosis of lung tumor has been attracted more interest. GABAB receptor ligand-directed nanoparticles consisting of baclofen functionalized trimethyl chitosan (Bac-TMC) as polymeric carriers, tripolyphosphate (TPP) as ionic crosslinker, and siSurvivin as therapeutic genes, were designed to enhance the survivin gene silencing. GABAB receptor agonist baclofen (Bac) was initially introduced into TMC as a novel ligand. This Bac-TMC/TPP nanoparticles increased the uptake of survivin siRNA through the interaction with GABAB receptor, further resulted in efficient cell apoptosis and gene silencing. For siRNA-loaded nanopartilcles pulmonary delivery, mannitol was utilized for it delivery into pressurized metered dose inhalers (pMDI).

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The fine particle fractions of this formulation was (45.39±2.99)% indicating the appropriate deep lung deposition. These results revealed that this pMDI formulation containing Bac-TMC/TPP nanoparticles would be a promising siRNA delivery system for lung cancer treatment.

Keywords: survivin siRNA; baclofen; trimethyl chitosan; nanoparticles; pressurized metered dose inhalers

Chemical compounds studied in this article Trimethyl chitosan (PubChem CID: none); Baclofen (PubChem CID: 2284); Sodium tripolyphosphate (PubChem CID: 24455); Mannitol (PubChem CID: 6251); HFA-134a (PubChem CID: 13129)

1. Introduction Small interfering RNA (siRNA) which can block the translation of specific mRNA and proteins by forming the RNA-induced silencing complex (RISC) after binding with ribozymes compounds, is considered a very promising therapeutic approach in the treatment of lung diseases. Survivin is a new member of the inhibitor of apoptosis (IAP) protein family with extremely anti-apoptotic effect (Sah, Khan, Khan, & Bisen, 2006), that over-expressed in most common human cancers including none small cell lung cancer (NSCLC) (Wang et al., 2016), while it is hardly expressed in normal cell/tissue. The effect of silencing survivin genes mediated by siRNA provide a promising modality in lung cancer therapy. Delivery of siSurvivin into lung caner cell could be achieved by intravenous injection or inhalation, the latter route is more advantageous than injection because it can avoid serum-induced aggregation and degradation by nuclease. Unfortunately, pulmonary delivery of siRNA faces some barriers including the mucociliary clearance action of the ciliated epithelial cells, the presence of mucus, phagocytosis by macrophages and intracellular barriers (Lam, Liang, & Chan, 2012). 2

To overcome the intracellular barriers, researchers have developed varieties of polymer carriers to facilitate the transfection efficiency of siRNA. Cationic chitosan-based carriers, such as poly(ethylene glycol)-grafted (PEGylated) chitosan (Rudzinski, Palacios, Ahmed, Lane, & Aminabhavi, 2016), amino acid modified chitosan (Ping et al., 2017) and peptide modified chitosan (Nascimento et al., 2017), have gained increasing attention owing to their low immunogenicity, biocompatibility and biodegradability compared with viral carriers (Saranya, Moorthi, Saravanan, Devi, & Selvamurugan, 2011). However, the poor water solubility and the low endocytic uptake of chitosan have posed a limitation for its use as gene delivery carriers. N,N,N-trimethyl chitosan (TMC), a cationized chitosan derivative, possesses good water solubility at physiological environment as well as excellent transfection efficiency due to its bind tightly to gene (Kean, Roth, & Thanou, 2005). TMC modified with many functional groups have been used for DNA/siRNA delivery exhibiting higher cellular uptake and target gene knockdown efficiency compared with chitosan carriers (Tang, Huang, Zhang, & Wang, 2014; Zhang, Tang, & Yin, 2013; Zheng, Tang, & Yin, 2015). Additionally, gene delivery carriers can efficiently convey genes to specific target tissues or cells with the aid of targeting ligands to recognize cancer cells, and then facilitate the internalization of gene. Ligands such as transferrin (Xie et al., 2016), folate (Shi, Zhang, Bi, & Dai, 2014), hyaluronic acid (Park, Noh, Kim, & Yong, 2017) and RGD peptides (Khatri, Rathi, Baradia, & Misra, 2014) have been used widely. Recently, some studies have shown that γ-aminobutyric acid type B (GABAB) receptors play an inhibitory role in pancreatic cancer (Banerjee, Al-Wadei, Al-Wadei, Dagnon, & Schuller, 2014; Schuller, Hussein, Al-Wadei, & Majidi, 2008a), hepatocellular carcinoma (Wang, Huang, & Chen, 2008), as well as lung cancer (Schuller, Al-Wadei, & Majidi, 2008b). Although GABA receptors are the major inhibitory neurotransmitter in the central nervous system, these receptors are also expressed in most non-neuronal tissues, including the lungs (Chapman, Hey, Rizzo, & Bolser, 1993). The expression of GABAB receptors (GABABR) is significantly higher in non-small cell lung cancers (NSCLC) tissues than the non-cancerous tissues (Zhang et al., 2013). GABABR-signaling strongly inhibits cAMP-dependent signaling in human small airway

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epithelial and pulmonary adenocarcinoma cells, further suppresses the phosphorylation of CREB and inhibits trans-activation of EGFR, thereby effectively impedes DNA synthesis, cell proliferation and cell migration (Schuller, et al., 2008b). These findings implicate that GABABR agonists may provide a novel targeted strategy to improve receptor-mediated gene delivery to lung cancer. This study developed GABAB receptor ligand-directed trimethyl chitosan (TMC) as siSurvivin carriers to enhance the uptake of A549 cells for efficient gene silencing, GABAB receptor agonist baclofen was initially introduced into TMC as a novel ligand, and we hope that this baclofen functionalized TMC (Bac-TMC) carriers can improve the lung tumor targeting and enhance endocytosis by the interaction with GABAB receptor. Pressurized metered dose inhalers (pMDI) are currently the most common way of inhalations for asthma or COPD treatment. As a revolutionary invention, pMDI possess great advantages, such as economically affordability and portability for patients, high-efficiency delivery, resistance to bacterial contamination and humidity, etc (Li, H. Y., & Xu, E. Y., 2017). Whereas they are facing great challenge of siRNA-carrier systerms formulated in the pMDI because the siRNA-loaded nanoparticles can not be compatible directly with propellant. Tremendous efforts have been made to resolve this problem. Sharma, Somavarapu, Colombani, Govind, & Taylor (2013) first explored HFA-227 based pMDI for crosslinked chitosan-based microparticles delivery, and they suggested this pMDI system have potential in delivery of nucleic acids. Subsequently, Conti, Brewer, Grashik, Avasarala, & Rocha (2014) developed a HFA-227 based pMDI for siRNA-loaded dendrimer complexes pulmonary delivery, which also exhibited favorable aerosol characteristics. Respiratory mucus could reduce the permeability of drugs, hinder the distribution of drugs in lung tissue. The use of mucus inhibitor, such as glycopyrrolate and mannitol, could improve the degree of hydration of mucus (Ferrari et al., 2001; Daviskas, Anderson, Eberl, & Young, 2008). To overcome the mucociliary clearance and mucus barrier, mannitol can be used as a component to prepare the pMDI with aerodynamic diameter betweent 1 μm and 5 μm for the siRNA pulmonary delivery system. In this paper, we explored the HFA-134a based pMDI formulation for pulmonary delivery of siRNA-loaded nanoparticles which has not been published in present study.

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To cross lung epithelial tissue barriers and intracellular barriers for siSurvivin pulmonary delivery, pMDI formulation containing siSurvivin-loaded baclofen functionalized TMC nanoparticles were thoroughly characterized in this paper. We evaluated the cytotoxicity, cellular uptake, and gene silencing efficiency of baclofen functionalized TMC nanoparticles. We further investigated the aerosolization properties of pMDI containing siSurvivin nanoparticles and successfully revealed that this siSurvivin-loaded nanoparticles pulmonary delivery can be realized by HFA-134a based pMDI. 2 Materials and methods 2.1 Materials N,N,N-trimethyl chitosan was purchased from Qihuadun Chemical Products Co. Ltd. (Zhengzhou, China). The molecular weight was 250 KDa determined by gel permeation chromatography, the degree of quaternization was to be 60% and the degree of deacetylation was to be 90% determined by 1H-NMR spectroscopy. Baclofen was purchased from Meilun Biotech Co. Ltd. (Dalian, China). Sodium tripolyphosphate (TPP) and mannitol were obtained from Aladdin Industrial Corporation (Shanghai, China). HFA-134a was purchased from Juhua Co. Ltd. (Zhejiang, China). The custom gene qRT-PCR quantitation kit, fluorescently labeled siRNA (FAM-siRNA), negative control siRNA (siNC, sense strand, 5’-UUC UCC GAA CGU GUC ACG UTT-3’), and siSurvivin (sense sequence, 5 ‘-GGA CCA CCG CAU CUC UAC AdTdT-3’) were synthesized by GenePharm (Shanghai, China). Other relevant reagents were supplied from KeyGen BioTech (Nanjing, China) and Aldrich Chemical. Co. (Shanghai, China). 2.2 Methods 2.2.1 Synthesis of Bac-TMC The TMC interacted with baclofen using carbodiimide chemistry was shown in Fig. 1. Taking the synthesis of Bac-TMC1 as an example, baclofen (0.25 mmol) and NHS/EDC (0.25/0.5 mmol) were added to 10 mL deionized water at pH 4.0 and stirred at 25℃ for 1 h. Then it was mixed with TMC (1 mmol). The reaction mixture was stirred at 20℃ for 24 h. The mixture was dialysed against deionized water (MWCO 3500) for 3 days and then lyophilized to

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obtain the final product. Bac-TMC2 and Bac-TMC3 were obtained by baclofen interacting with TMC at the molar ratio of 0.5:1 and 1:1, respectively.

2.2.2 Characterization of Bac-TMC UV-Vis spectrophotometer (Mapada UV-1800, China) was used to determine the substitution of baclofen in TMC backbone since baclofen has specific absorption peaks at 227 nm. According to the Beer-Lambert's law, the substitution degrees of the baclofen were decided based on the calibration curves. The structure of synthetic products were confirmed by FTIR spectrum (Tensor 27, Bruker, Germany) and 1H-NMR spectrum (Avance AV-III, Bruker, Switzerland). 2.2.3 Preparation and characterization of siRNA-loaded Bac-TMC nanoparticles The siRNA-loaded nanoparticles were formed spontaneously by the electrostatic interaction between cationic Bac-TMC with anionic TPP/siRNA. Detailedly, siRNA solution was mixed with TPP solution (0.5 mg/mL), then Bac-TMC solution (0.25 mg/mL) was added dropwise into the siRNA/TPP mixture under stirring to form siRNA-loaded nanoparticles (Bac-TMC/TPP/siRNA NPs). The weight ratio of Bac-TMC to TPP was fixed at 3:1. siRNA-loaded TMC nanoparticles (TMC/TPP/siRNA NPs) were prepared by the same method. The particle sizes and zeta potentials of different nanoparticles were determined using dynamic light scattering (DLS) by ZetasizerNano ZS (Malvern Instrument, UK). 2.2.4 Gel retardation assay The binding capability of siRNA to the TMC or Bac-TMC was evaluated by agarose gel electrophoresis using naked siRNA as a control. Various nanoparticles were prepared by mixing polymer (TMC, Bac-TMC1, Bac-TMC2, or Bac-TMC3) and 200 ng siRNA at different ratios from 1:1 to 20:1. The siRNA-loaded nanoparticles were kept at room temperature for 20 min before being mixed with 5×loading buffer. And then samples were loaded onto 1.5 wt% agarose gel in TAE running buffer and electrophoresed at 90 V for 20 min. The electrophoresis image was obtained by the gel imaging analysis system (Chemidoc XRS+, Bio-Rad, USA). 2.2.5 In vitro stability assay

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The stability of siRNA was examined by agarose gel electrophoresis. The Bac-TMC/TPP/siRNA nanoparticles were incubated with 10% fetal bovine serum (FBS) or bronchoalveolar lavage fluid (BALF) (1.0 mg/mL as protein concentration) at 37℃ for 24 h using naked siRNA as negative control. At every time internal, samples containing 200 ng siRNA were taken out. Before gel electrophoresis, heparin (10 mg/mL) was added into mixture to dissociate siRNA from nanoparticles. The siRNA integrity was investigated by the gel imaging analysis system. 2.2.6 Cytotoxicity assay A549 cells were seeded into 96-well microplate (5×103 cells/well) and cultured in RPMI 1640 medium supplied with 10% FBS and 1% penicillin/streptomycin for 24 h. Then the medium was replaced by 200 μL fresh medium containing different concentrations of TMC or Bac-TMC polymers (5-60 μg/mL), and then the cells were incubated for 48 h before 20 μL MTT (5 mg/mL) was added to incubate for another 4 h to measure cell viability. The medium was replaced by 150 μL of DMSO to dissolve the formed formazan crystal completely. Finally, the absorbance of formazan was measured directly using a Microplate Reader (Multiskan FC, USA) at the wavelength of 490 nm. Thus, cell viability was calculated as the ratio of the absorbance of treated cells to untreated cells. 2.2.7 In vitro cellular uptake In order to evaluate the transfection of nanoparticles, A549 cells were seeded in 12-well plates at a concentration of 105 cells/mL and cultured in medium overnight. The medium was then replaced by fresh serum-free medium which contained nanoparticles with 50 nM FAM-siRNA to transfect cells for 2, 4, 6 h. To reveal the role of baclofen residues in cellular uptake, free baclofen (100 μM) (Wang, et al., 2008) was added one hour prior to the addition of baclofen modified nanoparticles. The uptake efficiency of nanoparticles was quantified by the flow cytometer (MACSQuant Analyzer 10, Miltenyi, Germany). Furthermore, inverted fluorescence microscopy (Olmpeus IX53, Japan) was used to visualized observe the cells uptake. A549 cells were transfected with TMC/TPP/siRNA NPs or Bac-TMC/TPP/siRNA NPs for 4 h, fixed with 4% paraformaldehyde, and stained with Hoechst 33342.

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To investigate the uptake mechanism, A549 cells were transfected with FAM-siRNA loaded nanoparticles for 4 h at 4℃, or incubation with clathrin-mediated endocytosis inhibitors such as chlorpromazine (10 μg/mL), hypertonic sucrose (450 mM), or coveolae-mediated endocytosis inhibitors such as filipin (5 μg/mL), genistein (200 μM), or macropinocytosis inhibitior such as amiloride (13.3 μg/mL) for 30 min at 37 °C prior to the addition of the nanoparticles and throughout the 4-h uptake. 2.2.8 In vitro gene silencing and cell apoptosis assay To detect the gene silencing of nanoparticles loaded with siSurvivin, A549 cells were incubated in 6-plates with complete medium. On the following day, cells were treated with TMC/TPP/siSurvivin NPs or Bac-TMC/TPP/siSurvivin NPs in incomplete culture medium as test groups, while the nanoparticles loaded with siNC were set as the control groups. After 4 h transfection, the cells were further cultured for another 68 h with fresh free-serum medium. Then the total RNA were extracted from A549 cells using trizol reagent. 2 μg RNA was reverse transcribed into cDNA using the Custom gene qRT-PCR Quantitation Kit. The quantification of gene expression was detected with a real-time PCR system (LightCycler 96, Roche, Switzerland) in triplicate with a 20 μL volume. The percent knockdown from quantification cycle (Cq) values was obtained by quantitative real-time PCR (qRT-PCR) analysis in RNAi experiment. The gene expression data were analyzed using the comparative Cq (2-∆∆Cq) method. The ∆∆Cq relative gene expression values of survivin was normalized to the GAPDH endogenous control. The apoptosis of the siSurvivin-loaded nanoparticles was detected by Annexin V-FITC/PI apoptosis detection kit. A549 cells were seeded into 6-well plates and cultured until 70% confluence. Subsequently, the cells were treated with different nanoparticles (50 nM siSurvivin) for 4 h. siNC-loaded nanoparticles served as negative groups, TMC/TPP/siSurvivin NPs and Bac-TMC/TPP/siSurvivin NPs served as test group, meanwhile untreated cells group as blank control. Then the cells were cultivated with fresh medium for another 68 h. The apoptotic cells were detected by flow cytometry after staining with Annexin V-FITC and propidium iodide (PI).

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2.2.9 Preparation of the pMDI formulation Bac-TMC3/TPP/siRNA NPs were formed in deionized water as 2.2.3 described, and then the nanoparticles (equ. 125 μg siRNA ) were combined with mannitol giving the concentration of the excipient at 50 mg/mL or 100 mg/mL. The mixture was spray-dried (Mini Spray Dryer B-191, Buchi, Switzerland) using the following parameters: atomizing air flow = 473 L·h−1, aspiration = 70%, pump ratio = 5%, inlet temperature = 45°C, outlet temperature = 35°C. Dry mannitol microparticles containing siRNA-loaded nanoparticles were accumulated in the collection vessel for further experiments. To prepare pMDI formulation, a certain amount of mannitol microparticles was weighed into pressure proof glass vials (Shandong Pharmaceutical Glass Co., LTD, China) and crimp-sealed with a 63 μL metering valves (Aptar Pharma, USA). Then the propellant HFA-134a was added with the help of a propellant filling machine (Nanjing NJQWJ, China) and the concentration of microparticles was controlled at 2 mg/mL. The pMDI system was sonicated for 30 min to disperse microparticles completely in the propellant. 2.2.10 The physical stability and particle size The physical stability of the pMDI was investigated by the sedimentation rate experiment. When the sonication was stopped, digital images were taken to monitor the quality change of the dispersions at different time interval. The particle size of the mannitol microparticles was assessed by Mastersizer 2000 (Malvern Instrument, UK). Detailedly, microparticles loaded with nanoparticles were dispersed in 2H,3H perfluoropentane (HPFP) using a sonication bath. HPFP, which is liquid at ambient conditions, could as a model of propellent HFA (Rogueda, 2003). After HPFP evaporated, the microparticles were collected to measure the particle size. 2.2.11 The integrity of siRNA during spray drying and aerosolization After encountering the spray drying, the integrity of siRNA was assessed by densitometry. Briefly, an exact mass of particles were dissolved in 200 μL DEPC water and incubated at 4℃ to break down the mannitol shell. Then heparin was added and the siRNA content was quantified by densitometry based on gel electrophoresis. Naked siRNA which did not

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experience spray drying was regarded as negative control. The effect of propellant HFA-134a and the shear force during the aerosolization on the bioactivity of siRNA was also analyzed by gel electrophoresis. Several actuations were fired from the pMDI formulation, then microparticles were collected and stored in 4℃ with DEPC water. Next day, gel electrophoresis was proceeded before heparin was added into samples. 2.2.12 The aerosol characterization of pMDI To study the aerosolization of pMDI, FITC was conjugated to the free amine groups of Bac-TMC3 according to a modified method from previous study (Geisberger，Gyenge, Maake, & Patzke, 2013). Briefly, 3 mL of FITC (1 mg/mL) dissolved in methanol was added drop-wise to the equal volume of Bac-TMC3 aqueous solution (10 mg/mL) under stirring for 4 h in the dark at room temperature. Afterwards, the product was dried under vacuum to obtain yellow solid and then washed thoroughly with ethanol to remove any free FITC. FITC labeled Bac-TMC3 was used to prepare nanoparticles following the described in 2.2.3, and then the nanoparticles containing FITC labeled Bac-TMC was used to prepare pMDI as described in 2.2.9. The aerodynamic property of pMDI formulation was determined by the two-stage impinger (TSI) fitted with Chinese Pharmacopoeia induction and operated with a flow rate of (60±5) L/min. Before each TSI test, five actuations were fired to waste, subsequently 200 actuations were released into the TSI with an interval of 5 s between each actuation. Aerosol formulation in every stages was collected and rinsed thoroughly with a certain amount of DEPC water. The fluorescence intensity of FITC-labeled nanoparticles was measured at excitation and emission wavelengths of 492 nm and 518 nm using automatic microplate reader (POLARstar Omega, BMG, Germany). Fine particle fraction (FPF) was defined as the percentage of the content in Stage2 (mass median aerodynamic diameter < 6.4 μm) to the content of total samples released into the TSI. 3. Result and discussion 3.1 Characterization of Bac-TMC In order to improve the specificity and selectivity of siRNA-carrier delivery to lung cancer, 10

the baclofen groups were integrated to TMC polymers via the EDC/NHS mediated amidation reaction. In this study, the obtained polymers are named Bac-TMC1, Bac-TMC2, Bac-TMC3. From UV-Vis measurement based on the baclofen absorption at 227 nm, the percentages of baclofen substitution were found to be 4.71%, 12.74%, 22.27%, respectively. The FTIR spectra of TMC and Bac-TMC were shown in Fig. 2A. The characteristic bands of TMC polymer correspond to the stretching vibration of NH2 and OH combined peak (3,413 cm−1), C=O stretching vibration bond of the acetamido groups (1,640 cm−1), N-H bond of the amino group (1,564 cm−1), and C-H bond of methyl groups (1,480 cm−1). Comparing with TMC, the amide Ⅱband (1568 cm−1) was remarkable in the spectrum of Bac-TMC, indicating the formation of anamide bond after the baclofen conjugation. The new peaks located at 1151 cm-1 and 800 cm-1~950 cm-1 belonged to the in-plane and out-plane bending vibration of benzene ring, and the presence of benzene was expected in the region of 7.09-7.37 ppm in the 1H-NMR spectrum of Bac-TMC (Fig. 2B), further indicating baclofen groups were successfully conjugated to the backbone of TMC.

3.2 Characterization of siRNA-loaded nanoparticles The partcle size determines the uptake efficiency of siRNA delivery system. It has been previously reported that particles in the range of 50 nm to several hundred nanometers are effectively internalized into cancer cell (Song, Sun, Zhang, Zhou, & Zhang, 2008; Liu & Reineke, 2005). As shown in Table 1, the stable nanoparticles with minimum sizes of about 210 nm were able to form when the optimal weight ratio of TMC to TPP was 3:1. The particle sizes of the nanoparticles increased slightly from 211 nm to 232 nm with the increasing of modified degree of baclofen groups, which was ascribed to the stereo hindrance effect of baclofen groups. The zeta potential determines the stability of nanoparticles, influences the cell cytotoxicity due to the interaction with negative charge of the cell membrance, even more effects the transfection efficiency of siRNA-loaded nanoparticles. As shown in Table 1, the zeta potential of particles was negative at smaller ratios of TMC to TPP. In this condition, TPP and siRNA

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were competitive binding with TMC, making siRNA unable to be loaded in particles. Apparently, the particle size of nanoparticles was more than 360 nm when the zeta potential was neutral or negative. In this condition, the particles tend to aggregate resulting in the increase in particle size. Subsequently, zeta potentials turned to positive when the ratio of TMC/TPP exceed 2:1. The zeta potentials of nanoparticles formed by TMC or Bac-TMC were varied from +20.11 mV to +22.92 mV at the polymer/TPP ratio of 3, indicating that the nanoparticles were positively charged. It has been reported that the zeta potential values between ±20-30mV usually means the moderately stability of nanoparticles (Patel & Agrawal, 2011).

3.3 Gel retardation assay siRNA binding ability is a significant property when developing gene delivery system. As shown in Fig. 3A, the siRNA was completely retarded at the polymer/siRNA weight ratio of 5:1 to 20:1, demonstrating TMC/TPP and Bac-TMC/TPP possessed strong binding capacity with siRNA. TMC, the trimethyl derivant of chitosan, has higher water solubility and positively charge possessing prominent siRNA loading effciency than chitosan. The introduction of TPP into chitosan-based nanoparticles could enhance the siRNA binding capacity owning TPP carriers five negative charges, consequently increase the efficiency of siRNA-mediated gene silencing (Xiao et al. 2017; Zhang, et al. 2013). 3.4 In vitro stability assay In order to investigate the protection ability of the nanoparticles, Bac-TMC3/TPP/siRNA was chosen as a model nanoparticles to incubate with serum or BALF for 24 h. As Fig. 3B shown, naked siRNA was hardly detected after incubation with serum for 2 h., while the integrity of siRNA within nanoparticles was maintained about 77.34% under the protection of the polymer depending on the image analysis technique of optical density. Similarly, the integrity of siRNA in BALF was also enhanced under the protection of nanoparticles compared with naked siRNA. Importantly, siRNA was more stable in lung than serum indicating the superiority of pulmonary delivery.

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3.5 In vitro cytotoxicity of the nanoparticles Cationic polymers potentially induce cell cytotoxicity at a certain level since these positive charged groups interact with negative charged proteins on the cell surface. Thus, the viability of cells treated with different polymers was tested by MTT assay in vitro. The cell viability results were displayed in Fig. 4A, PEI 25 kDa showed high cytotoxicity even at a low concentration of 5 μg/mL. Compared with PEI, the cell viability was much higher in both TMC groups and Bac-TMC groups. On average, cell viability was over 80% when A549 cells were treated with the four different polymers for 48 h at the concentration of 5 μg/mL, while the viability was decreased when the cells were co-incubated with polymers at relatively high concentration, suggesting that TMC-based polymers exhibited concentration-dependent cytotoxicity. The cell viability declined with the increasing of polymers was a result of an increase in the net positive charge. Whats more, it should be noticed that the cell viability of Bac-TMC polymers groups was lower than TMC group, especially when the cells were incubated with Bac-TMC polymers at the concentration of 20 μg/mL, the cell viability fell to about 50%~70%, and when the concentration of Bac-TMC polymers increased to 40 μg/mL, the cell viability was only 27% approximately. This result confirmed that baclofen could reduce the DNA synthesis and cell proliferation of small airway-derived adenocarcinoma lung (Schuller, 2008b). According to the optimal concentration of siRNA (50 nM-100 nM) in vitro experiment, the cell viability still exceeded 70% after incubation with polymers at the 10:1 polymer-siRNA ratio. 3.6 Cellular uptake study In this article baclofen was employed originally as a novel ligand expecting it directed towards GABAB receptor overexpressing in lung cancer cells. The rationale of using baclofen was to reduce non-specific toxicity to normal cells and increase the gene knockdown mediated by siRNA. As shown in Fig. 4B, naked FAM-siRNA was barely internalized into cells, while cells were incubated with different nanoparticles displayed excellent uptake efficiency, especially those incubated with Bac-TMC2/TPP/siRNA NPs and Bac-TMC3/TPP/siRNA NPs for 4 h. As seen in Fig. 4C, siRNA which delivered by Bac-TMC3/TPP NPs experienced a 2.6-fold uptake

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level in A549 cells compared with that none baclofen functionalized nanoparticles following 4 h incubation (P<0.01), sustaining their cellular entry was enhanced by baclofen groups. The uptake of Bac-TMC3/TPP/siRNA NPs significantly decreased to 40.51% after cells co-incubated with free baclofen (100 μM), suggesting that the uptake of Bac-TMC/TPP/siRNA NPs was blocked due to free baclofen coupling with GABAB receptors, this results further confirmed that the active entry of baclofen functionalized nanoparticles into A549 cells was mainly mediated by ligand-recepor interaction. It is worth mentioning that the uptake of Bac-TMC3/TPP/siRNA NPs at 6 h was almost same compared with the uptake at 4 h, while the uptake of other nanoparticles was promoted when the cells were kept incubating to 6 h. This result implied the nanoparticles with high substituted degree of baclofen groups can not only increase the uptake of siRNA, but also shorten the uptake period. Fluorescence images showed a more straightforward result of cellular uptake of siRNA incubated with differents nanoparticles at 4 h (Fig. 5). Baclofen functionalized nanoparticles displayed stronger fluorescence in the cytosol compared with TMC/TPP/siRNA NPs, which was consistent with the quantitative data measured by flow cytometry. It has been reported that the ligand-receptor interaction mostly falls into clathrin-dependent endocytosis (Khalil, Rathi, Baradia, & Misra, 2006). Therefore we investigated the cellular uptake pathway of siRNA-loaded nanoparticles. TMC and Bac-TMC3 were chosen as model polymers. As seen in Fig. 4D, the cellular uptake of Bac-TMC3/TPP/siRNA NPs was decreased more than 96% incubating at 4°C while TMC/TPP/siRNA NPs was only decreased to 85%, which implied that Bac-TMC3 was more dependent on energy-mediated endocytosis (P <0.001). However, unchanged internalization level was found following treatment with amiloride indicated that the cellular entry of polymers/TPP/siRNA NPs was irrelevant to macropinocytosis pathway. Comparatively, the internalization of TMC/TPP/siRNA NPs was predominantly via clathrin-dependent and coveolae-mediated endocytosis. The uptake of Bac-TMC3/TPP/siRNA NPs was greatly enhanced by clathrin-mediated pathway (P<0.05) indicated the taken up of Bac-TMC3 nanoparticles predominantly via clathrin-dependent pathway owing to the ligand-receptor

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mediated endocytosis.

3.7 Gene silencing and cell apoptosis analysis Survivin gene was employed as the therapeutic target in this study due to its well-established contribution to tumorous proliferation. To evaluate silencing efficiency of siSurvivin-loaded nanoparticles, real-time quantitative PCR was performed by monitoring the mRNA level. As shown in Fig. 6A, Bac-TMC2/TPP/siSurvivin NPs and Bac-TMC3/TPP/siSurvivin NPs significantly reduced the mRNA expression of survivin gene to (52.94±3.60)% and (35.32±2.68)%, respectively, while the gene expression of unfunctionalized nanoparticles was reduced to (62.89±6.27)% (p<0.001). As the negative control, the polymers/TPP/siNC NPs didn’t show significant silencing effect on survivin expression. To further examine whether baclofen functionalized nanoparticles enhance apoptosis or not, A549 cells were double stained by Annexin V-FITC/PI after treatment with naked siSurvivin, siNC-loaded nanoparticles and siSurvivin-loaded nanoparticles. As depicted in Fig. 6B, in comparison with TMC/TPP/siSurvivin NPs group which only induced (28.12+1.25)% apoptosis, Bac-TMC2/TPP/siSurvivin NPs group and Bac-TMC3/TPP/siSurvivin NPs group induced (42.81±0.52)% and (57.89±3.10)% apoptosis cells respectively (n=3, p<0.001). As discussed in the cellular uptake experiment, the introduction of baclofen groups into TMC molecules increased the uptake ability of siRNA, further induced the higher gene silencing of Bac-TMC/TPP/siSurvivin nanoparticles against survivin gene than TMC/TPP/siSurvivin nanoparticles, consequently resulted in much higher apoptosis of A549 cells. According to the gene silencing and cell apoptosis analysis, Bac-TMC could be regarded as a promising siRNA carrier to down-regulate the expression of target gene. 3.8 Preparation and characterization of nanoparticles loaded pMDI It has been reported that optimum aerodynamic particle size for deep lung deposition is between at 1-5 μm (Agu, et al, 2001; Sakagami, 2006). But the particle size of Bac-TMC3/TPP/siRNA NPs was 232 nm that far from the requirement. Besides, the other

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challenge is the physical stabilization of the microparticles dispersing in the low dielectric propellant HFA (Peguin, Wu, & Rocha, 2007; Wu, Bharatwaj, Panyam, & Rocha, 2008). In this study, we develop spray-dry granulation technology to form mannitol microparticles containing nanoparticles. Mannitol particles have shown suitability for deep lung deposition and ability to minimize the uptake by alveolar macrophages. Furthermore, mannitol, as an mucus inhibitor, can be used to overcome the mucus barrier (Ferrari et al., 2001). In our preliminary experiments, the mannitol microparticles dispersed stably in HFA-134a than lactose, trehalose and sodium carboxymethylcellulose (CMCC-Na) (data not shown). Young et al. (2009) also have reported that the physical stability of mannitol particles is much higher than other sugar particles suspending in HFA-134a propellant. The diameter of mannitol microparticles containing Bac-TMC3/TPP/siRNA NPs was characterized by dry disperse method after microparticles were removed from HPFP, and the results were shown in Fig. 7(A and B). The mean diameter of mannitol microparticles was found to be 6.53 ± 0.09 μm (sample 1) and 3.64 ± 0.06 μm (sample 2), respectively. These results suggest that the microparticles prepared by mannitol at the concentration of 100 mg/mL have good aerodynamic properties for appropriate deep lung deposition than sample 1 which was prepared by mannitol at the concentration of 50 mg/mL. Besides, it is worth noticing that the particle sizes of the nanoparticels loaded in microparticles were 229.0 ± 24.5 nm (sample 1) and 234.0 ± 20.7 nm (sample 2) after encountering the formulation process, which demonstrates that the engineering process of forming the microparticles and pMDI formulation did not seem to induce any change of the particle sizes of the nanoparticles (P<0.05).

3.9 Physical stability of microparticles in HFA-134a The physical stability of the mannitol microparticles containing the Bac-TMC3/TPP/siRNA NPs in HFA-134a was evaluated through sedimentation rate experiments. As insets in Fig. 7 (A and B) presented, the microparticles (50 mg/mL mannitol) were aggregated immediately in HFA-134a when stopping the mechanical sonication, and whole particles aggregated on the walls of the canister after 2 h settling time, while there were only observed some aggregation of microparticles at the pMDI containing (100 mg/mL 16

mannitol). And in the high concentration of mannitol, the aggregates formed due to sedimentation could be easily redispersed by manual shaking of the canister. Researches have been reported that the physical stability of microparticles suspending in HFA-227 had greater physical stability than HFA-134a (Cocks, Somavarapu, Alpar, & Greenleaf, 2014; Sharma, et al., 2012). This may be attributed to the greater density of HFA-227 being closer to that of the microparticles compared to HFA-134a as a result of formulation prepared in HFA-134a facing up tough challenge. This study indicated that the combination of mannitol into nanoparticles can enhanced the physical stability of HFA-134a based pMDI system. 3.10 The integrity of the siRNA after spray drying and aerosolization The integrity of the siRNA after undergoing spray drying and aerosolization was evaluated by gel electrophoresis assays and further analyzed using the ImageJ software (Fig. 7C). The integrity of siRNA was decreased to be 66%~67% of the original integrity after spray drying of the siRNA-loaded nanoparticles to form microparticles and then the integrity was slightly decreased to be 60~63% of the original integrity when the microparticles was aerosolized from HFA-134a based pMDI. The optical density of the siRNA was found to be very similar between the two different groups, indicating the siRNA was degraded mainly due to the thermal energy generating from the process of spray drying. siRNA can be successfully loaded into mannitol microparticles to overcome the shear force of aerosolization process and conserve its integrity. 3.11 The aerosolisation property of the pMDI The FITC labeled Bac-TMC3 was used to quantify the formulation concentrations of microparticles in different stages of TSI after aerosolization. There was not statistical difference between the particle sizes of FITC-labeled nanoparticles and the unlabeled nanoparticles (p>0.05, data not shown). Then the pMDI was prepared as previous method by FITC labeled Bac-TMC3/TPP/siRNA NPs. The microparticles of every stages were collected from TSI with DEPC water to calculate FPF. As shown in Fig. 7D, the FPF of sample 1 and sample 2 was (20.65±6.57)% and (45.39±2.99)%, respectively (p>0.05). The high deposition of the later microparticles delivered

17

from pMDI indicated suitable aerosolization properties for pulmonary delivery, while the former microparticles were aggregated immediately in propellant resulting in lower lung deposition. This results suggested that mannitol microparticles loaded with siRNA nanoparticles which dispersed homogeneously in HFA-134a could serve as potential candidate for siRNA pulmonary delivery. This pMDI system loaded with Bac-TMC/TPP/siRNA NPs possesses highly siRNA binding ability, efficiently gene silencing and proper aerosolisation performance in lung environment. Conclusion In this study, we confirmed that baclofen groups introduced into TMC could increase the uptake ability of siRNA-loaded nanoparticles through the interaction with GABAB receptor, ultimately resulting the gene silencing and cell apoptosis effectively. We successfully encapsulated Bac-TMC3/TPP/siRNA NPs into mannitol microparticles for its dispersion in HFA-134a based pMDI formulation which possessed good aerodynamic properties for deep lung deposition. According to our research, pulmonary delivery of siRNA can avoid the serum-induced degradation. In addition, the efficiency of gene silencing and cell apoptosis induced by siSurvivin was improved in lung cancer cell due to the target recognition of Bac-TMC/TPP nanoparticles. These study suggested that this pMDI formulation containing Bac-TMC/TPP/siRNA nanoparticles could overcome lung epithelial tissue barriers and intracellular barriers, which would be a promising siRNA pulmonary delivery system for lung cancer treatment.

Acknowledgments This work was supported by the 3rd Jiangsu Overseas Research & Training Program for University Prominent Young & Middle-aged Teachers and Presidents; the College Students Innovation Project for the R&D of Novel Drugs [No. J1310032]. And we would like to thank cell and molecular biology experiment platform of China Pharmaceutical University for the assistance with relevant test items.

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Pharmaceutical Research, 32(6), 2038-2050.

Figure captions Fig. 1. Synthetic routes of baclofen functionalized trimethyl chitosan (Bac-TMC). Fig. 2. (A) The FTIR spectra of TMC and Bac-TMC. (B) The 1H-NMR spectra of TMC and Bac-TMC. Fig. 3. (A) Gel retardation analysis of siRNA binding with TMC/TPP NPs or Bac-TMC/TPP NPs. (B) Gel electrophoresis assays the stability of siRNA in serum and BALF. Fig. 4. (A) Comparison on cytotoxicity of carriers tested with A549 cells, (n=6). (B) Internalization identification of nanopartilces in A549 cells with flow cytometric analysis. (C) Quantification of cell internalization was shown by mean fluorescence intensity (MFI), Relative uptake level(%) = MFI (treated group)/MFI (control group) ×100%. (*P<0.1, **P<0.01, ***P<0.001). (D)

Effects of endocytosis inhibitors and temperature on the internalization of siRNA loaded nanoparticles (n=3). Fig. 5. Fuorescence images about cellular uptake of FAM-siRNA within TMC/TPP NPs and Bac-TMC/TPP NPs for 4 h. A549 cells were treated with TMC/TPP/siRNA NPs (A), Bac-TMC1/TPP/siRNA NPs (B), Bac-TMC2/TPP/siRNA NPs (C), Bac-TMC3/TPP/siRNA NPs (D), respectively. (E) A549 cells were pretreated with free baclofen (100 μM) and then treated with Bac-TMC3/TPP/siRNA NPs. Images showed fluorescent overlay of siRNA (green, FAM-labeled) and nuclei (blue, Hoechst33342-stained). Fig. 6. (A) siSurvivin mediated silencing of A549 was assessed using qRT-PCR (∆∆Cq), GAPDH as endogenous reference gene, (n=3). (B) Flow cytometry analysis for apoptosis induced by different nanoparticles, (n=3). Fig. 7. Particle sizes and physical stability (in insets) of sample 1 microparticles (50 mg/ml mannitol) (A) and sample 2 microparticles (100 mg/ml mannitol) (B). (C) Gel electrophoresis assays of the integrity of siRNA within mannitol microparticles, (n=3). (D) Aerosol properties of pMDI formulations prepared with Bac-TMC3/TPP/siRNA NPs loaded into mannitol microparticles, (n=3).

23

Fig. 1

Fig. 2

24

Fig. 3

25

Fig. 4

26

Fig. 5

27

Fig. 6

28

Fig. 7

Table 1. Particle size and zeta potential of TMC/TPP/siRNA NPs and Bac-TMC/TPP/siRNA NPs with the weight ratios of polymers/siRNA at 10:1. Data were presented as mean ±SD (n =3). Polymer/TPP Polymer

Size(nm)

PDI

Zeta potential (mV)

(w/w) TMC

1.5:1

374.6±7.2

0.091±0.013

-18.81±0.49

TMC

2:1

363.4±12.3

0.327±0.043

0

TMC

2.5:1

267.6±15.4

0.347±0.082

+18.42±0.69

TMC

3:1

209.0±5.0

0.194±0.025

+22.46±2.32

TMC

4:1

448.3±42.5

0.876±0.223

+39.81±3.34

TMC

3:1

211.3±4.07

0.199±0.054

+20.11±1.33

Bac-TMC1

3:1

220.4±6.40

0.240±0.021

+22.92±1.11

Bac-TMC2

3:1

226.7±9.35

0.219±0.053

+21.54±1.83

29

Bac-TMC3

3:1

232.1±7.11

30

0.141±0.022

+20.78±2.94