Targeted tumor delivery and controlled release of

Journal of Controlled Release 232 (2016) 131–142

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Journal of Controlled Release journal homepage: www.elsevier.com/locate/jconrel

Targeted tumor delivery and controlled release of neuronal drugs with ferritin nanoparticles to regulate pancreatic cancer progression Yifeng Lei a, Yoh Hamada b, Jun Li a, Liman Cong b, Nuoxin Wang a, Ying Li a, Wenfu Zheng a,⁎, Xingyu Jiang a,⁎ a

Beijing Engineering Research Center for BioNanotechnology, CAS Key Laboratory for Biological Effects of Nanomaterials and Nanosafety, CAS Center for Excellence in Nanoscience, National Center for NanoScience and Technology, Beijing 100190, China Department of Nano-Medical Science, Graduate School of Medicine, Tohoku University, Sendai 980-8575, Japan

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a r t i c l e

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Article history: Received 21 October 2015 Received in revised form 17 February 2016 Accepted 14 March 2016 Available online 2 April 2016 Keywords: Ferritin nanoparticles Drug delivery Pancreatic cancer Nervous microenvironment

a b s t r a c t Pancreatic cancer is a lethal malignancy whose progression is highly dependent on the nervous microenvironment. This study develops neural drug-loaded ferritin nanoparticles (Ft NPs) to regulate the nervous microenvironment, in order to control the pancreatic cancer progression. The drug-loaded Ft NPs can target pancreatic tumors via passive targeting of EPR effects of tumors and active targeting via transferrin receptor 1 (TfR1) binding on cancer cells, with a triggered drug release in acidic tumor environment. Two drugs, one activates neural activity (carbachol), the other impairs neural activity (atropine), are encapsulated into the Ft NPs to form two kinds of nano drugs, Nano-Cab NPs and Nano-Ato NPs, respectively. The activation of the nervous microenvironment by Nano-Cab NPs significantly promotes the pancreatic tumor progression, whereas the blockage of neural niche by Nano-Ato NPs remarkably impairs the neurogenesis in tumors and the progression of pancreatic cancer. The Ftbased nanoparticles thus comprise an effective and safe route of delivery of neural drugs for novel anti-cancer therapy. © 2016 Elsevier B.V. All rights reserved.

1. Introduction Pancreatic cancer is one of the most lethal solid malignancies with a 5-year survival rate of less than 5% [1]. Even if treated with surgery and the standard first-line chemotherapeutic drug, Gemcitabine (GEM), the median overall survival of patients is only 7.2 months [1]. Thus, new therapeutic approaches are urgently needed to treat this lethal disease. Emerging evidence suggests that therapies targeted at nervous microenvironments provide a novel means to regulate the progression of certain cancers [2–7]. In particular, nervous microenvironment is a crucial factor during the early invasive growth and metastatic spread of pancreatic cancers [8]. The process of perineural invasion (PNI), in which tumor cells grow and spread along native nerve fibers, represents a prominent pathologic feature of pancreatic cancer [9]. PNI is considered the foremost reason for poor patient survival, high tumor recurrence and severe neuropathic pain in pancreatic cancers [9,10]. Thus, targeting nervous microenvironment and associated

⁎ Corresponding authors. E-mail addresses: [email protected] (W. Zheng), [email protected] (X. Jiang).

http://dx.doi.org/10.1016/j.jconrel.2016.03.023 0168-3659/© 2016 Elsevier B.V. All rights reserved.

neural signaling may have novel potentials in pancreatic cancer treatment. Herein in this study, we aim to target the nervous microenvironment in order to regulate the progression of pancreatic cancers, by target the most important nerve–cancer interaction. The pancreas receives abundant innervations from the autonomic nervous system, including sympathetic and parasympathetic nervous system (Scheme S1) [11, 12]. Acetylcholine (ACh) is one of the most important neurotransmitters in the autonomic nervous system [13]. ACh released from nerve endings acts on muscarinic acetylcholine receptors (mAChRs) on stromal cells of the pancreas (Scheme S1), controlling the physiological function of pancreas [14]. Emerging evidences indicate that muscarinic signaling (ACh–mAChR interaction) plays important roles in cancer progression [2,3,15,16]. mAChRs are expressed on pancreas stromal cells, pancreatic cancer cells and pancreatic carcinoma [17,18]. These observations suggest that targeting the nervous microenvironment by muscarinic signaling has great potential in the regulation of pancreatic cancer progression. Herein, we aim to investigate the effects of muscarinic signaling on pancreatic cancer progression, by applying muscarinic agonist, carbachol, and muscarinic antagonist, atropine, to activate or block the neural niche in pancreatic cancers, respectively. These drugs are clinically approved therapeutics and are already in routine clinical use [19].

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Y. Lei et al. / Journal of Controlled Release 232 (2016) 131–142

However, the systemic administration of neural drugs may evoke potential side effects similar to the effects of a nerve agent [19], thus the tumor site-specific drug delivery of these neural drugs is essential. Different delivery systems have been constructed to promote targeted drug delivery using nanotechnology, including liposomes, polymer nanoparticles, and nanogels [20,21]. Among them, ferritin nanoparticles (Ft NPs) provide an effective platform for tumortargeted delivery and drug release [22–24]. Ferritin is a spherical protein composed of 24 subunits of heavy and light chains which forms a hollow nanocage [25]. Ft NPs possess a great biocompatibility and safety profile, because they exist naturally in the human body [26–28]. Ferritin nanocage can encapsulate a large amount of small drug molecules [23,27,29]. Moreover, ferritin composed only of heavy chain can bind to tumors via transferrin receptors (TfR, TfR1, or CD71) [30], which are overexpressed on many types of cancer cells. These unique properties make Ft NPs ideal delivery vehicles for tumor-targeting in our study. Herein, we aim to use the Ft NP platform to deliver neural drugs (carbachol, atropine) to pancreatic tumor sites, in order to regulate the tumor nervous microenvironment, and study their effects on pancreatic cancer progression. The selection of Fn NPs as neural drug carrier for pancreatic cancers comes from the consideration of the alkaline nature of pancreatic juice in the body (pH = 8.2) [31]. The Ft NPs will only release drugs in the mildly acidic environment in pancreatic tumor tissues, without leaking drugs during the delivery process (including the circulation in blood and in pancreatic juice). Thus, we are able to study the effects of local regulation of tumor nervous microenvironment on the progression of pancreatic cancers (Scheme 1). Our results show that the Ft NP-based neural drug delivery is efficient in regulating the nervous microenvironment, and as a consequence, regulating the neurogenesis and the progression of pancreatic cancers (Scheme 1), suggesting great potential of targeting nervous microenvironment of tumors for novel anti-cancer therapy.

2. Materials and methods 2.1. Materials and reagents Ferritin, Gemcitabine hydrochloride (GEM), and inorganic reagents were from Sigma-Aldrich. Doxorubicin was obtained from Dalian Meilun Biotech, China. Carbamoylcholine chloride (carbachol) and atropine were purchased from Abcam. Cell culture medium and complements were from Invitrogen unless indicated. 2.2. Preparation of drug-loaded ferritin NPs The loading of drugs into the cavity of ferritin (Ft) was prepared with the process of denaturation and renaturation of protein [32]. Ferritin at 1 mg/mL was dissolved in 8 M urea and gently mixed for 30 min at room temperature to ensure complete dissociation of the ferritin. Each type of drug (including atropine, carbachol, and doxorubicin) was added into the solution with a final concentration of 1 mg/mL. After incubation for 15 min in darkness, the mixture was transferred into dialysis bags (MWCO 3500 Da, Thermo Scientific) and dialyzed against gradient concentrations of urea buffer (7, 5, 3, 2, 1 and 0 M) containing 1 mg/mL of respective drug at 4 °C to slowly reassemble Ft nanocages (Fig. 1A). After Ft refolding, the drug-loaded Ft NPs were dialyzed against saline overnight to remove the free drugs and non-specific binding of drugs to Ft NPs. The Ft NPs encapsulated with atropine, carbachol and doxorubicin were labeled as Nano-Ato NPs, Nano-Cab NPs and Nano-Dox NPs, respectively. 2.3. Characterization of drug-loaded NPs The size, polydispersity index (PDI), and zeta-potential of the prepared Ft NPs were characterized by dynamic light scattering (DLS, Zeta Sizer Nano ZS, Malvern Instruments Ltd.). The analysis was

Scheme 1. Illustration of neural drug-loaded Ft NPs for regulation of pancreatic cancer progression.


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Fig. 1. Preparation and characterization of drug-loaded ferritin nanoparticles (Ft NPs). (A) Schematics of the process of drug encapsulation into Ft nanocages. (B–F) TEM images of natural Ft nanocages, disassembled Ft with urea, and re-assembled Ft encapsulated with atropine (Nano-Ato NPs), carbachol (Nano-Cab NPs) and doxorubicin (Nano-Dox NPs), respectively. The inserts are higher magnification images. (G) TEM analyses of the sizes of Ft NPs during the preparation. (*0.01 b p b 0.05).

performed at 25 °C and the concentration of Ft was 0.1 mg/mL in PBS buffer. For transmission electron microscopy (TEM), nanoparticle suspension was deposited onto carbon-coated copper grids, and observed by TEM (Tecnai G2 20 S-TWIN, 200 kV).

2.4. Determination of drug content The Ft concentration in Ft NPs was determined using a BCA protein assay kit (Beyotime). The concentration of atropine was determined by HPLC (Waters 2796) using a Luna C18 column (250 × 4.6 mm, 5 μm, Phenomenex) with mobile phase of 0.5 M potassium dihydrogen-acetonitrile (82:18) and detected at 210 nm. And the concentration of carbachol was determined by HPLC using mobile phase of 40 mM ammonium acetate-acetonitrile (30:70) and detected at 210 nm. The concentration of doxorubicin was determined by measuring the absorbance at 485 nm using a calibration curve of a series of doxorubicin dilution. The drug:Ft molar ratio was calculated accordingly.

2.5. Diameter changes of Ft NPs against pH conditions The Ft NPs were dissolved in pre-determined buffer solution with different pH conditions, including acetate buffer (0.1 M, pH 5.0 and pH 5.5), sodium phosphate buffer (0.1 M, pH 6.5 and pH 6.8), PBS buffer (0.1 M, pH 7.4), and sodium bicarbonate buffer (0.1 M, pH 8.2). After incubation at 37 °C for 12 h, 24 h, and 48 h, the hydrodynamic diameter and size distribution of the Ft NPs were characterized by DLS, the morphology of the Ft NPs at the dried state was observed by TEM.

2.6. In vitro drug release To test the drug release from the Ft NPs, 500 μl of the Nano-Dox NP samples (500 μM Dox equivalent) was placed in D-tube (MWCO 6–8 kDa, Novagen) and dialyzed in solution under varying pH conditions (acetate buffer (0.1 M, pH 5.0), sodium phosphate buffer (0.1 M, pH 6.5), PBS buffer (0.1 M, pH 7.4), and sodium bicarbonate buffer (0.1 M, pH 8.2)) at 37 °C in darkness. After different incubation time, 1 mL aliquots of the medium were withdrawn, and replaced with 1 mL of fresh medium. The released Dox at different incubation times was determined by HPLC using mobile phase of water-acetonitrileacetic acid (80:19:1) and detected at 485 nm. The accumulative release of Dox from Nano-Dox NPs was expressed as a percentage of the released drug and plotted as a function of time. 2.7. Cell culture The human Panc-1 pancreatic cancer cells were obtained from Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, and cultured in DMEM medium containing 10% fetal bovine serum (FBS) and 1% penicillin streptomycin (PS). Panc-1 cells stably transfected with luciferase gene (Panc-1-luc cells) were obtained from Institute of Laboratory Animal Sciences, Chinese Academy of Medical Science, and maintained in DMEM supplemented with 10% FBS, 1% PS and 600 μg/mL G418. The cell lines were routinely maintained in a humidified atmosphere containing 5% CO2 at 37 °C. 2.7.1. Panc-1 cellular uptake of Ft NPs For cellular uptake of Ft NPs, Nano-Dox NPs were used for visualization due to the intrinsic fluorescence of Dox (excitation 485 nm,

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emission maximum 595 nm). Briefly, Panc-1 cells were seeded at a density of 1 × 105 cells/cm2 onto confocal dish (Nunc, diameter 20 mm). After 24 h incubation, the medium were removed and cells were washed with Hanks' Balanced Salt solution (HBSS). The cells were incubated with free Dox and Nano-Dox (10 μg/ml Dox equivalents in HBSS) at 37 °C for 1 h and 3 h, respectively. After incubation, the cells were washed three times with PBS, fixed with 4% paraformaldehyde (PFA), cell nuclei were counterstained with DAPI. The cell samples were mounted in Prolong® Gold antifade reagent (Invitrogen) and observed with confocal microscopy (Carl Zeiss LSM710) with 488 nm laser excitation to observe the fluorescence signal of Dox. 2.7.2. Antibody blocking assay To confirm that the TfR1 is the binding receptor of Ft NPs to pancreatic cancer cells, an antibody blocking study was carried out by incubating the Panc-1 cells with Nano-Dox NPs in the presence of an excess of anti-TfR1 mAb (Abcam). The Nano-Dox NPs were diluted with a Dox concentration and Ft concentration to be 10 μg/mL and 249 μg/mL (0.565 μM), respectively. A solution of anti-TfR1 antibody (5.65 μM) was incubated with the Nano-Dox NPs for the competitive binding of TfR1 on the Panc-1 cells. The cells were observed by confocal microscopy and compared with the cells incubated in the absence of TfR1 mAb.

2.8. Animal studies All animal experiments were performed with the approval of the Institutional Animal Care and Use Committee, Institute of Process Engineering, Chinese Academy of Sciences (IACUC, IPE, CAS, IRB Number 2014-0002). Balb/c nude mice (male, 8 week, ~ 20 g) were obtained from Vital River Laboratory Animal Center (Beijing, China) and raised under SPF conditions.

2.8.1. Circulation half-life of Ft NPs in blood To determine the circulation half-life of Ft NPs in blood, free Dox or Nano-Dox NPs (10 mg/kg Dox equivalents) was intravenously injected into healthy Balb/c nude mice (n = 4 per group). At different time points post-injection, blood from the tail vein of mice was collected in heparin-treated tubes and the blood plasma was separated. The Dox from plasma was extracted by acidified isopropanol, and the concentration of Dox was determined by measuring its fluorescence intensity at 485 nm excitation and 595 nm emission on an EnSpire Multimode Plate Reader (PerkinElmer). To correct the nonspecific background, the fluorescence of blood samples from untreated mice was determined and deducted from each measurement. The concentration of Dox in each group was calculated with a calibration curve of a series of Dox dilution. The results were plotted as the Dox concentration as a function of post-injection time, and the circulation half-life (t1/2) was calculated with the Drug and Statistics (DAS) software.

2.8.2. Biodistribution and tumor targeting of Ft NPs in tumor-bearing mice For bio-distribution analysis of Ft NPs in tumor-bearing mice, NanoDox NPs were used due to the intrinsic fluorescence of Dox. Balb/c nude mice were injected with 2 × 106 Panc-1 cells into the subcutaneous place of upper flank of the to form subcutaneous tumors. When the tumor sizes reached about 0.5 cm in diameter, the mice were intravenously injected with free Dox and Nano-Dox (10 mg/kg Dox equivalents) (n = 4 per group). At various time points post-injection, in vivo fluorescence images of mice were acquired with Maestro™ 2 in vivo imaging system (CRi) with the green filter (from 560 nm to 750 nm stepped in 10 nm increments) with constant exposure time of 1200 ms. For ex vivo fluorescence imaging, mice were killed, tumors and the major organs of mice were collected and visualized with the same system.

Table 1 Drug and drug concentration used in orthotopic pancreatic tumors. Drugs

Functions

Concentration (drug equivalents)

Carbachol Nano-Cab Atropine Nano-Ato Saline Gemcitabine

Muscarinic agonist Ft NPs with carbachol encapsulation Muscarinic antagonist Ft NPs with atropine encapsulation Control Chemotherapeutic drug for pancreatic cancer

0.25 mg/kg 0.25 mg/kg 0.30–0.45 mg/kg 0.30–0.45 mg/kg 150 mmol/L 20 mg/kg

2.8.3. Effect of neural drug-loaded Ft NPs on pancreatic tumor progression Orthotopic pancreatic tumor models were created to mimic the natural pancreatic cancer microenvironment. Briefly, Balb/c nude mice were anesthetized. A mid-line incision was made in the lower abdomen of the mice, and approximately 2 × 106 Panc-1-luc cells in 20 μl PBS were injected into the pancreas head of the mice using a Hamilton microsyringe. The incision was closed by a running suture of 5–0 silk. Two weeks after the tumor cell implantation, the mice were randomly sorted into different groups (n = 6–9 per group), and treated with different drug formulations as listed in Table 1, via tail vein injection in 100 μl volume for each formulation. The drug concentration in the Ft NPs was determined by HPLC before each injection, and the drug administration was carried out twice a week for 4 weeks. The tumor growth was monitored by bioluminescence imaging (BLI), mouse body weights were measured during the experiment. Mice were monitored for up to 42 days post-implantation and then sacrificed. Primary tumors, spleen and mesenteries were harvested for ex vivo imaging and immunofluorescence analyses. 2.8.4. Bioluminescence imaging The orthotopic pancreatic tumor growth was monitored by bioluminescence imaging (BLI, Xenogen IVIS Spectrum). Bioluminescent signal was induced by intraperitoneal (i.p.) injection of 150 mg/kg D-luciferin (Promega) into the mice bearing Panc-1-luc tumors 8–10 min before the in vivo imaging. Mice were anesthetized using 1.5% isofluorane during whole imaging process. Imaging time ranges from 10 s to 5 min, depending on the bioluminescence intensity of the primary tumors or metastatic lesions. For ex vivo imaging, 150 mg/kg D-luciferin were injected 8 min prior to necropsy, animals were sacrificed and organs of interest were immersed in saline containing 300 μg/ml D-luciferin, and imaged for 1 to 2 min. 2.9. Histology and immunofluorescence Upon euthanasia of animals, the pancreatic tumor tissues were processed in OCT fixation (Tissue-Tek) for histological evaluation. Frozen sections of the tumor tissues (5 μm) were stained by standard hematoxylin and eosin procedure (H&E). For immunofluorescence staining, frozen sections of the tumor tissues (5 μm) were fixed with 4% PFA, incubated in 3% H2O2 to quench endogenous peroxidase, permeabilized with 0.3% Triton X-100, and blocked by 10% goat serum. For nerve identity in tumor tissues, sections were incubated with a chicken pAb to neurofilament heavy (NF-H, Millipore) and a rabbit pAb to neurofilament light (NF-L, Millipore) at 4 °C overnight, followed by secondary antibody of goat anti-chicken Alexa Fluor® 488 and goat anti-rabbit Alexa Fluor® 555 (Invitrogen) at 37 °C for 1 h, respectively. Extracellular matrix of tumor tissues were incubated with a rabbit pAb to collagen I (Abcam), followed by Alexa Fluor® 555-conjugated goat antibody to rabbit IgG (Invitrogen). For blood vessels staining, samples were incubated with a rabbit pAb to CD31 (Abcam) and then Alexa Fluor® 555-conjugated goat antibody to rabbit IgG. For proliferative cell quantification, sections were incubated with a rabbit pAb to Ki67 (Abcam), followed by Alexa Fluor® 555-conjugated goat antibody to rabbit IgG. Cell nuclei were counterstained with DAPI at room temperature for


10 min. The samples were mounted with antifade reagent and observed with confocal microscopy. To quantify the nerve density, ten independent fields (0.045 mm2) within the sections of NF-L and NF-H staining were randomly selected, NF-L and NF-H staining were color-merged and transformed into 8-bit images with ImageJ software, and the overall nerve density was evaluated by counting the nerve areas per field by setting the same threshold.

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Table 3 Characterization of drug content in Ft NPs.

Nano-Ato NPs Nano-Cab NPs Nano-Dox NPs

Ft concentration (μg/mL)

Drug concentration (μg/mL)

Drug:Ft molar ratio

475.5 825.7 648.0

14.6 20.0 26.0

46.7:1 58.3:1 32.5:1

2.10. Western blotting The expression of transferrin receptor 1 (TfR1) by Panc-1 cells, the expression of nerve growth factor (NGF) in pancreatic tumors were assessed by Western blot assay. Lysates of cells or tumors were prepared with lyses buffer, separated on 10% SDS-polyacrylamide gel, and transferred to a PVDF membrane. The membrane was blocked with 5% non-fat milk, 0.1% Tween 20 in PBS for 30 min at room temperature, and incubated with primary antibody, including mouse anti-TfR1 mAb (Abcam) or rabbit anti-NGF mAb (Abcam) at 4 °C overnight, followed by secondary antibody conjugated to horseradish peroxidase (HRP) (Jackson). The Western blot signals were detected using an enhanced chemiluminescence system (Amersham, GE Healthcare), and the intensity of proteins was quantified using a Bio Imaging System. 2.11. Statistical analysis All in vitro experiments were based on at least three independent samples for each condition, and the experiments were repeated for three times. The in vivo experiments were performed with 4 to 9 mice for each condition. Data were presented as mean ± standard deviation (s.d). Statistical analysis of the samples was performed using student's t-test, and p value less than 0.05 was considered statistically significant. 3. Results 3.1. Preparation and characterization of drug-loaded Ft NPs We prepared the Ft NPs loaded with the muscarinic antagonist, atropine (Nano-Ato NPs), and with the muscarinic agonist, carbachol (Nano-Dox NPs), respectively. We also prepared the Ft NPs encapsulated with doxorubicin (Nano-Dox NPs) where doxorubicin has an intrinsic fluorescence thus helps for the visualization. The loading of drugs into the cavities of Ft nanocages was carried out via the disassembly and reassembly of Ft (Fig. 1A). Natural Ft proteins showed nanocage structures (Fig. 1B). Ft proteins broke down into subunits after denaturation with 8 M urea (Fig. 1C). After gradual dialysis, the Ft fragments refolded and formed Ft NPs encapsulated with atropine (Nano-Ato NPs), carbachol (Nano-Cab NPs) and doxorubicin (Nano-Dox NPs), respectively (Fig. 1D–F). TEM and DLS analyses showed that each kind of drug-loaded Ft NPs was well dispersed and had uniform sizes (Table 2). The sizes of drug-loaded Ft NP were larger than the original Ft NPs (Fig. 1G, Fig. S1, Table 2), most likely due to the successful encapsulation of drugs into the cavity of the Ft nanocages. We characterized the drug content in each kind of Ft NPs. The concentrations of Ft in the Nano-Ato, Nano-Cab and Nano-Dox NPs were determined by BCA protein assay, which were 475.5, 825.7, and 648.0 μg/mL, respectively (Table 3). The concentrations of atropine, carbachol, and doxorubicin in the Nano-Ato, Nano-Cab and Nano-Dox

NPs were measured to be 14.6, 20.0 and 26.0 μg/mL using HPLC and calibration curves, respectively (Table 3). Accordingly, the molar ratios of Ato:Ft, Cab:Ft and Dox:Ft were calculated to be 46.7:1, 58.3:1 and 32.5:1, respectively (Table 3). 3.2. Drug release from Ft NPs We systematically studied the diameter changes of the Ft NPs against typical pH conditions (Fig. S2). After 24 h incubation at 37 °C, the hydrodynamic diameters of the Ft NPs were almost constant around 14 nm by DLS at both pH 8.2 and 7.4 (Fig. S2A, C). The hydrodynamic diameter of the Ft NPs increased to 68.4 nm at pH 6.5, and significantly increased at pH 5.0 (Fig. S2A, C). To further confirm the size variation of Ft NPs in different pH conditions, we evaluated the morphology of the Ft NPs with TEM (Fig. S2B). The Ft NPs had a dehydrated diameter around 12.0 nm at pH 8.2 and pH 7.4 after incubation at 37 °C for 24 h (Fig. S2B, C). In contrast, the dehydrated diameter of Ft NPs slightly increased to 13.7 nm at pH 6.5, and significantly increased to 27.7 nm at pH 5.0 (Fig. S2B, C). These results suggest that the diameters of the Ft NPs can increase in response to the slight pH difference between normal pancreas tissues and tumor tissues. We investigated the drug release from the Ft NPs by HPLC method. The drug release were evaluated at pH 8.2, 7.4, 6.5, and 5.0, which simulated the normal pancreatic juice condition (pH 8.2), the blood stream and normal physiological environment (pH 7.4), the mildly acidic environment in tumor tissues (pH 6.5), and the acidic lysosomes (pH 5.0), respectively [31,33,34]. We found that the Ft NPs disassembled into protein subunits under acidic conditions and released the encapsulated molecules both time- and pH- dependently (Fig. 2). The Ft NPs were relativelt stable in pH 8.2 condition (mimicking pancreas liquid) and in pH 7.4 condition (mimicking blood circulation), with less than 15% of the drugs was released from Ft NPs over 60 h of incubation (Fig. 2). In contrast, the acidic environment significantly increased the release rate of drugs from Ft NPs (Fig. 2). At pH 5.0 and 6.5, drug release was found to be 86% and 61% by 60 h of incubation, respectively. 3.3. In vitro cellular uptake of Ft NPs mediated by TfR1 binding We investigated the cellular uptake of Ft NPs by Panc-1 cells using the intrinsic fluorescence of Dox (Fig. 3). After 1 h and 3 h incubation, a larger number of Nano-Dox NPs accumulated into the Panc-1 cells. In contrast, little amount of free Dox was observed in the Panc-1 cells. To demonstrate the active tumor-targeting ability of the Ft NPs by TfR1 binding, we carried out competition binding experiments using TfR1 antibody. The TfR1 expression on Panc-1 cells was confirmed by western blotting assay (Fig. 3A). There was a significant decrease of the accumulation of Nano-Dox NPs into Panc-1 cells after the blockage

Table 2 Characterization of drug-loaded Ft NPs during synthesis.

NP size by TEM (nm) NP size by DLS (nm) PDI Zeta-potential (mV)

Ft nanocage

Ft unfolding

Nano-Ato NPs

Nano-Cab NPs

Nano-Dox NPs

11.71 ± 0.35 11.96 ± 1.35 0.33 ± 0.05 −7.14 ± 0.67

– 18.71 ± 1.97 0.64 ± 0.12 −27.78 ± 2.56

12.28 ± 0.49 13.08 ± 1.24 0.34 ± 0.08 −6.66 ± 1.15

12.31 ± 0.45 13.54 ± 1.31 0.27 ± 0.05 −6.65 ± 1.92

12.27 ± 0.47 13.26 ± 1.43 0.38 ± 0.09 −6.55 ± 1.86

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Fig. 2. In vitro drug release profiles from the Nano-Dox NPs in buffer at pH 5.0, 6.5, 7.4, and 8.2, which simulate the acidic lysosomes, the mildly acidic environment of solid tumor tissues, blood circulation and normal physiological condition, and the normal pancreatic juice, respectively.

of TfR1 (Fig. 3B). The quantitative analysis showed that the average fluorescence intensity in the Nano-Dox treated cells was about 3-fold greater than those treated with free Dox group and the group of Nano-Dox NPs with TfR1 blocking (Fig. 3C). 3.4. Circulation half-life of Ft-NPs in vivo To study the pharmacokinetics of Ft NPs, we intravenously injected Nano-Dox NPs and free Dox into healthy Balb/c nude mice, and studied their circulation half-life by analyzing the Dox concentration in the blood plasma at different time post-injection (Fig. 4A). The measured blood elimination half-life of Nano-Dox NPs was 289 ± 43.5 min. In contrast, free Dox showed a quick washout from the circulation, and the blood half-life of free Dox was only 16.8 ± 3.6 min (Fig. 4A). The significantly extended half-life of Nano-Dox NPs in blood is beneficial to improving drug retention in the systemic circulation and facilitates the accumulation of drugs to tumor sites. 3.5. In vivo bio-distribution of the Ft NPs We investigated the biodistribution and tumor targeting ability of Ft NPs in tumor-bearing mice using the intrinsic fluorescence of Dox

(Fig. 4B, C). Balb/c nude mice bearing subcutaneous Panc-1 tumors were intravenously injected with Free Dox and Nano-Dox NPs. With the increase of time, significantly more Nano-Dox NPs accumulated into the tumor sites than free Dox, and the fluorescence signal of Dox in the tumor tissues was increased over time (Fig. 4B, D). After 6 h of i.v. injection, we sacrificed the mice and collected the major organs and tumors for ex vivo fluorescent imaging (Fig. 4C). We found high intensity of fluorescence in the kidney (Fig. 4C). The reason for this may come from two aspects: 1) The kidney is an excretory organ where free Dox and Nano-Dox NPs could be filtrated and excreted in the urine, thus it is not surprising to observe high intensity of Dox fluorescence in the kidney. 2) The kidney is one of the major organs of the reticuloendothelial system (RES), considering the uptake and retention of drug and nanoparticles by the RES, our data were in accordance with previous reports of the accumulation of drugs or Ft NPs in the kidney [27,35]. In contrast, we found the fluorescence signal of Nano-Dox NPs at the tumor sites was significantly higher than that of free Dox (Fig. 4C), and the fluorescence intensity of Nano-Dox NPs at the tumor sites was 3-fold of that of free Dox by quantitative region-of-interest analysis (Fig. 4E). The enhanced tumor retention of Nano-Dox NPs could be attributed to the combination of prolonged circulation halflife of Ft NPs (Fig. 4A), the enhanced permeability and retention (EPR) effect of tumors [36], and the active targeting mechanism via TfR1 binding on cancer cells [30] (Scheme 1). Together, the enhanced tumor accumulation of Ft NPs (Fig. 4), and acidic tumor microenvironmenttriggered drug release from Ft NPs (Fig. 2, Fig. S2) are beneficial for improving the targeted delivery and drug release at the pancreatic tumor sites. 3.6. Orthotopic tumor progression We investigated the effect of neural drug-load Ft NPs onto the progression of pancreatic cancers. We implanted Panc-1-luc cells into the head of pancreas to form orthotopic tumors in mice, to mimic tumor microenvironment that most resembles the in vivo scenario. We monitored the tumor growth by bioluminescence imaging (BLI), where bioluminescence was a result of the luciferase of Panc-1-luc cells acting on D-luciferin (injected into the mice prior to imaging). Tumors grew rapidly in mice treated with muscarinic agonist of free Cab and Nano-Cab, similar to the saline control group (Fig. 5A,C). Nano-Cab enhanced tumor growth more effectively than free Cab (Fig. 5A,C). Free Cab induced a decreased body weight and survival rate of mice to 33% on day 42 post-implantation (Fig. 5E,F), which can

Fig. 3. Cellular uptakes of Ft NPs mediated by TfR1 on Panc-1 cells. (A) Western-blotting of TfR1 expression on Panc-1 cells and in control cells of human umbilical vein endothelial cells (HUVECs). (B) Fluorescence images of Panc-1 cells incubated with free Dox, Nano-Dox in the absence or presence of a 10-time molar excess of anti-TfR1 mAb for 1 h and 3 h, respectively. Cell nuclei were counterstained with DAPI (blue). (C) Quantification of fluorescence (FL) intensity of Dox in (B).


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Fig. 4. Blood circulation half-life and tumor targeting of Ft NPs in vivo. (A) Dox concentration in mouse plasma as a function of time post-injection of free Dox and Nano-Dox NPs (10 mg/kg Dox equivalent) into healthy Balb/c nude mice (n = 4). (B) Whole body fluorescence images of Dox in Balb/b nude mice bearing subcutaneous Panc-1 tumors at 1 h and 6 h after tail vein injection of free Dox and Nano-Dox NPs, respectively. After 6 h, free Dox was distributed throughout the body, Nano-Dox was selectively accumulated in the tumor region. The tumors were marked by white circles. (C) Ex vivo fluorescence images of major organs and Panc-1 tumors at 6 h after i.v. injection of free Dox and Nano-Dox NPs, respectively. (D) In vivo quantification of the total fluorescence intensity of Dox in the Panc-1 tumor sites at 1 h and 6 h, respectively. (E) Ex vivo quantitative biodistribution of the total fluorescence intensity of Dox in major organs and tumors at 6 h after i.v injection. (*p b 0.01).

be attributed to the systemic toxicity of carbachol [19]. Nano-Cab NPs reduced the systemic toxicity of carbachol and increased the survival rate of mice to 83% on day 42 (Fig. 5E, F), suggesting the advantages of Ft NPs for an effective and safe route of neural drug delivery. On the other hand, the administration of muscarinic antagonist of free Ato and Nano-Ato suppressed the tumor growth, similar to the chemotherapeutic drug GEM-treated mice (Fig. 5A, C). The Nano-Ato led to more significant reduction of tumor growth than free Ato group (Fig. 5A, C), with survival rate of 100% for administration of both Nano-Ato and free Ato on day 42 (Fig. 5E, F). The systemic administration of chemotherapeutic drug GEM was toxic to mice and induced a rapid decrease of body weight and survival rate of the mice to 0% on day 34 (Fig. 5E, F). Ex vivo evaluation showed that the muscarinic agonists promoted, whereas muscarinic antagonists suppressed the tumor growth and metastasis to the spleen and mesenteries (Fig. 5B, D).

3.7. Immunofluorescence analysis We performed the histological analysis of pancreatic tumor tissues by H&E staining (Fig. S4A). The Nano-Cab NP treated tumor was well maintained in structure and better than the saline control, whereas the Nano-Ato NP treatment induced obvious necrosis in tumor tissues (Fig. S4A), suggesting that Nano-Ato treatment induced active antitumor effect.

We evaluated the nervous microenvironment of the pancreatic tumor specimens with immunofluorescent staining to the neuronspecific cytoskeletal subunits of neurofilament-H (NF-H) and neurofilament-L (NF-L), which marked the mature neurons and immature neurons, respectively [37]. Both NF-H and NF-L neurons were new born nerves in the tumor tissues, and most nerves contained both NF-H and NF-L staining (Fig. 6A,B, Fig. S3). The results suggested that the pancreatic tumor recruited the formation of new nerves (neo-neurogenesis) with different maturity (mature and immature neurons) within the tumor tissues. Interestingly, we found that the degree of total neurogenesis of tumor specimens were positively correlated with the extent of pancreatic cancer aggressiveness: Nano-Cab NPs promoted tumor growth, with significantly increased nerve density within tumor areas and in pancreas stromal tissues surrounding the tumors, as compared to the saline control group (Fig. 6A–C, Fig. S3). In contrast, Nano-Ato NP treatment inhibited the tumor progression with a reduced nerve density in the pancreatic tumors and peritumoral tissues (Fig. 6A–C). Moreover, the nerve density in pancreatic tumors was positively correlated with the expression level of nerve growth factor (NGF) protein in the isolated tumors (Fig. 6D). We also performed the immunofluorescence staining of pancreatic tumor sections with collagen I, CD31, and Ki67 antibody, which stained collagen network (an important component in the extracellular matrix), blood vessels and the proliferative cells, respectively (Fig. S4B–

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D). Tumor treated with Nano-Cab NPs showed a dense and interconnected collagen network surrounding tumor cells. In addition, tumor angiogenesis (formation of blood vessels) and the cancer cell proliferation were significantly enhanced by the Nano-Cab NP treatment as compared to saline control (Fig. S4B–D). In contrast, Nano-Ato NPs resulted in a weak and disrupted collagen network in tumor tissues, and the number of blood vessels and proliferative cells in tumors were significantly lowered by Nano-Ato treatment as compared to saline control (Fig. S4B–D). 4. Discussion Emerging evidence suggests that nervous microenvironment plays critical role in the progression of certain cancers [2–6]. Herein, we demonstrated that targeting nervous microenvironment via Ft NPs-based drug delivery can regulate the pancreatic cancer progression (Scheme 1). Mild unfolding-refolding process of Ft proteins was used to load neural drugs into Ft nanocages (Fig. 1A). The loading of drugs into the Ft nanocages is mainly dependent on electrostatic interactions. The internal surface of Ft is negatively charged in the neutral loading buffer [25], making it convenient for positively charged, small-molecule drugs to be loaded into the cavity of Ft nanocages. The pKa of atropine, carbachol and doxorubicin is 9.6 [38], 12.5 [39], and 8.2 [40], respectively, they can easily bind to the internal surface of Ft in the neutral loading buffer. The amount of encapsulated drugs in Ft NPs was calculated to be 47 atropine, 58 carbachol and 32 doxorubicin molecules per Ft nanocage (Table 3), which was correlated with the pKa value of these drugs: the higher the pKa of the drug, the more the drugs are loaded into the Ft cages. DLS and TEM analyses demonstrated the process of drug encapsulation into the Ft NPs (Fig. 1, Fig. S1). The sizes of the drugencapsulated Ft NPs were larger than natural Ft NPs (Table 2), most likely due to the successful drug encapsulation into Ft NPs. In previous studies, Ft NPs were developed to deliver the chemotherapeutic anti-cancer drugs to tumors, such as doxorubicin, which interacts with DNA and stops the DNA replication of cancer cells, thus to inhibit cancer progression [23]. Be differ from traditional strategies, herein we focus on the targeting of nervous microenvironment in pancreatic cancers. Ft NPs were used to effectively deliver the neural drugs to pancreatic tumor sites, thus to regulate the nervous microenvironment and the progression of pancreatic cancers (Scheme 1), which may represent a novel anti-cancer strategy. The drug release profile of Ft NPs indicated that no substantial drug release was detected in buffer at 8.2 and 7.4 even after 60 h incubation (Fig. S2, Fig. 2). In comparison, in acidic condition at pH 6.5 and 5.0, the Ft NPs released the encapsulated drugs (Fig. S2, Fig. 2), most likely due to the disassembly of the Ft NPs. The partial disassembly of the ferritin at pH 6.5 may lead to the formation of aggregates of the disassembled protein (ferritin in disordered states), the lower pH condition (pH 5.0) may lead to more aggregates formation due to the increase of the disassembled ferritin. Thus the particle size in the DLS became very large under a low pH conditions. The in vitro drug release profile suggested that drug-loaded Ft NPs were stable during the systemic delivery process in blood stream (pH 7.4) and in pancreatic juice (pH 8.2) [31], whereas the drug-loaded Ft NPs can release drugs in mild acidic environment of pancreatic tumors (pH 6.5) and further in the lysosomes of tumor cells (pH 5.0) [33,34]. Previous studies showed that Ft NPs composed of heavy chain ferritin specifically targeted tumor cells that overexpress TfR1 [30].

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Interestingly, TfR1 is also a marker of human pancreatic cancers [41]. We confirmed the expression of TfR1 on Panc-1 cells by western blotting (Fig. 3A). Antibody blocking assays confirmed the accumulation of Ft NPs to Panc-1 cells via TfR1 binding (Fig. 3). The in vivo experiments also validated the prolonged circulation half-life of Ft NPs in blood stream, and enhanced accumulation of the Ft NPs to pancreatic tumor sites due to the EPR effect of tumors and TfR1 binding of cancer cells (Fig. 4). Together, the enhanced accumulation of Ft NPs to tumor sites and the acidic pH-sensitivity of the Ft NPs are advantageous for neural drug delivery to tumor environment: during circulation in the bloodstream (pH 7.4) and in normal pancreatic juice (pH 8.2) [31], the encapsulated drugs were not released prematurely into the blood-stream and in normal pancreas. After reaching the pancreatic tumor sites by EPR effect and TfR1 binding, the encapsulated neural drugs can be released from the Ft NPs due to the acidic tumor environment [33,34], and effectively change the nervous microenvironment of pancreatic tumors (Scheme 1). Herein, we are interested in how the regulation of nervous microenvironment by Ft NPs contributed to pancreatic cancer progression. We encapsulated the agonist of muscarinic receptors, carbachol, and the antagonist of muscarinic receptors, atropine, into the Ft NPs (Scheme 1), to activate or block the neural niche of tumors, and evaluated their effects on Panc-1-luc tumor developments. Nano-Cab NPs significantly stimulated tumor growth and metastases, with decreased toxicity to mice as compared with free Cab (Fig. 5). In contrast, the Nano-Ato NPs suppressed the tumor progression more effectively than free Ato (Fig. 5). These results indicate: i) the Ft NPs are efficient drug carriers for specific tumor targeting, the nano-drug functioned more effectively than free drugs, proving the excellent property of Ft-based nanoparticle system for effective and safe drug delivery; ii) the regulation of muscarinic signaling by local delivery of neural drugs controlled the progression of pancreatic cancers efficiently. Our observations were in accordance with previous investigations of the effects of muscarinic signaling on other cancer models, for example, the knockdown of mAChR gene inhibited prostate cancer progression in mice [2], muscarinic agonist stimulated, whereas muscarinic antagonist inhibited the gastric tumorigenesis through mAChR-mediated Wnt signaling [3], and muscarinic antagonists inhibited cell growth in vitro and decreased tumor growth in vivo in small cell lung carcinoma [42]. These findings support that mAChR antagonists may be clinically useful for the treatment of cancers. To understand the mechanism of nervous microenvironment in pancreatic cancer progression, we investigated the nerve density and expression of NGF protein in tumor tissues. NGF is a neurotrophin most important for the growth and maintenance of nerves, the NGF/ TRKA (tropomyosin receptor kinase A) signaling is critical for the nerve infiltration, and perineural invasion of pancreatic cancers [9,43– 46]. We found that the pro-cancer effect of Nano-Cab NPs, and the anti-cancer effect of Nano-Ato NPs were correlated with the appearance and density of nerves in orthotopic tumors (Figs. 5,6), and the nerve density was positively correlated with the NGF levels in the tumors (Fig. 6). Our observations demonstrated the neurotropism of pancreatic cancer, which is consistent with previous clinical observations where pancreatic tumor specimens had a higher expression of the NGF and enhanced neural density as compared to normal pancreas [12,45,47,48]. The significant pro-cancer effects of Nano-Cab, and anticancer effects of Nano-Ato could be attributable to efficient cooperation of Ft

Fig. 5. Regulation of pancreatic tumor progression by neural drug-loaded Ft NPs. Panc-1-luc cells were implanted into pancreas head to form orthotopic tumors in mice on day 0, mice were treated with free Cab, Nano-Cab, free Ato, Nano-Ato, saline and GEM from day 15 post-tumor implantation. (A) In vivo bioluminescence images illustrated the pancreatic tumor progression in mice on day 14, 21, 31, and 38 post-implantation of orthotopic tumors. Mice treated with GEM died at day 34, and no mice were imaged thereafter. (B) Ex vivo images of bioluminescent signals in pancreatic tumors, and in excised spleen and mesenteries with metastases from indicated mouse groups, the pancreas was marked by yellow lines, pancreatic tumors were indicated by yellow arrow head, and the spleen was labeled as “S”. The specimen for GEM group was from the mice imaged on day 31, others were from day 42. (C) Serial in vivo bioluminescence analyses evaluated the growth of orthotopic pancreatic tumors. The black arrows indicated the day of administration of neural drugs. (D) Quantification of bioluminescence intensity of tumor metastases in excised spleen and mesenteries. (E) Animal survival curves in different groups. (F) The effect of different treatments on the body weight of mice (n = 6–9 per group). (*p b 0.01, **0.01 b p b 0.05).

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Fig. 6. Neural drug-loaded Ft NPs regulate the nervous microenvironment in pancreatic tumors. (A) Nerve distribution in the stroma surrounding solid pancreatic tumors, (B) Nerve infiltration in pancreatic tumor tissues at the end of in vivo experiment on day 42. Neural identity was confirmed by staining with the neuro-specific cytoskeletal subunits of neurofilament-L (NF-L, red), and neurofilament-H (NF-H, green), which marked immature neurons and mature neurons, respectively. The developing NF-L+ branches arising from a double-positive nerve fibers give nerves in yellow color (merged red and green). (C) Quantification of nerve areas positive for NF-L and NF-H in peritumor tissues and in pancreatic tumors, respectively. Each counted field surface =0.045 mm2. (D) Western-blotting of the expression of nerve growth factor (NGF) in solid pancreatic tumors at the end of in vivo experiments. (*p b 0.01).

NP-mediated local drug delivery and activated muscarinic signaling in pancreatic tumors (Fig. 7). In the tumor microenvironment, the local delivery and drug release of carbachol from Nano-Cab NPs activated

Fig. 7. Regulation of neuron-pancreatic tumor crosstalk with ferritin NPs to control pancreatic cancer progression. Neurons release acetylcholine (ACh), which acts on muscarinic receptor (mAChR) on pancreatic cancer cells. The targeted tumor delivery and local drug release of Nano-Cab NPs in the tumor microenvironment activates the ACh–mAChR pathway, leads to PI3K/AKT/mTOR mediated transcription and increased tumor growth, promoting the neurogenesis in tumors and PNI of tumors. In contrast, the delivery of Nano-Ato NPs inhibits the tumor growth, neurogenesis and PNI by disrupting the neural-tumor crosstalk.

the muscarinic signaling of tumor cells (Fig. 7), which stimulated the downstream signaling involving such as PI3K/AKT/mTOR pathway [16], resulting in gene translation, cancer cell survival, growth, proliferation, and invasion [49]. The growth of pancreatic tumors secreted several neurotrophic factors (such as NGF), attracting nerves surrounding the tumors and inducing the neo-neurogenesis in tumors (Fig. 6, Fig. S3) [50]. In turn, these tumor-innervating nerves released neurotransmitters (such as ACh) that trigger specific receptors on cancer cells, boosting the tumor growth, and the perineural space also provided a positive microenvironment for cancer spreading and metastasis (Fig. 7). These mutual attractions and reciprocal interactions resulted in both tumor development and nerve invasion of pancreatic carcinoma (Figs. 5, 6, Fig. S4) [51]. In contrast, the local delivery and drug release of atropine from Nano-Ato NPs blocked the muscarinic signaling in the tumor microenvironment, resulting in the impaired tumor growth and reduced neurogenesis in pancreatic tumors (Figs. 5, 6, Fig. S4). Our results revealed that the blockage of neuronal signaling with muscarinic antagonists effectively inhibit the pancreatic cancer progression (Figs. 5–7). Together, our data revealed a mutual tropism and reciprocal interaction between the neurogenesis and pancreatic cancer progression, which is analogous to angiogenesis in tumor progression [52]. Targeted drug delivery by Ft NPs resulted in increased bioavailability of the neural drugs at pancreatic tumor sites, with reduced side effects and amplified drug activity. Although future studies are required to dissect the molecular bases linking tumor neurogenesis to pancreatic cancer progression, our data proved that antagonists of neural signaling have great promise to inhibit pancreatic cancer progression. Acetylcholine is one of the


most important neurotransmitters implicated for pharmacotherapy, and multiple muscarinic antagonists are already used in routine clinics with minimal side effects [53], thus the clinical barriers for their use as novel cancer therapeutics should be relatively low, suggesting a great potential of these neural drugs to cancers. 5. Conclusion We successfully developed the neural drug-loaded Ft NP systems to regulate the nervous microenvironment and progression of pancreatic cancers. The activation of nervous microenvironment by Nano-Cab NPs significantly enhanced the pancreatic tumor growth and metastasis, whereas the blockage of neural niche by Nano-Ato NPs remarkably suppressed the neurogenesis in tumors and the pancreatic tumor progression. Our study suggests that Ft NP-based neural drug delivery is efficient in regulating nervous microenvironment, and has great potential in delivering other neural drugs for anti-cancer therapy. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.jconrel.2016.03.023. Acknowledgments We thank Professor Jane Y. Wu at Northwestern University for critical discussions. This work is supported by CAS (XDA09030305), NSFC (81361140345, 31470911, 31500775). Y.F.L. is supported by China Postdoctoral Science Foundation (2014M550672) and the Scientific Research Foundation for the Returned Overseas Chinese Scholars, State Education Ministry. References [1] A. Stathis, M.J. Moore, Advanced pancreatic carcinoma: current treatment and future challenges, Nat. Rev. Clin. Oncol. 7 (2010) 163–172. [2] C. Magnon, S.J. Hall, J. Lin, X. Xue, L. Gerber, S.J. Freedland, P.S. Frenette, Autonomic nerve development contributes to prostate cancer progression, Science 341 (2013) 1236361. [3] C.M. Zhao, Y. Hayakawa, Y. Kodama, S. Muthupalani, C.B. Westphalen, G.T. Andersen, A. Flatberg, H. Johannessen, R.A. Friedman, B.W. Renz, A.K. Sandvik, V. Beisvag, H. Tomita, A. Hara, M. Quante, Z. Li, M.D. Gershon, K. Kaneko, J.G. Fox, T.C. Wang, D. Chen, Denervation suppresses gastric tumorigenesis, Sci. Transl. Med. 6 (2014) 250ra115. [4] C.S. Scanlon, R. Banerjee, R.C. Inglehart, M. Liu, N. Russo, A. Hariharan, E.A. van Tubergen, S.L. Corson, I.A. Asangani, C.M. Mistretta, A.M. Chinnaiyan, N.J. D'Silva, Galanin modulates the neural niche to favour perineural invasion in head and neck cancer, Nat. Commun. 6 (2015) 6885. [5] S.C. Peterson, M. Eberl, A.N. Vagnozzi, A. Belkadi, N.A. Veniaminova, M.E. Verhaegen, C.K. Bichakjian, N.L. Ward, A.A. Dlugosz, S.Y. Wong, Basal cell carcinoma preferentially arises from stem cells within hair follicle and mechanosensory niches, Cell Stem Cell 16 (2015) 400–412. [6] H.S. Venkatesh, T.B. Johung, V. Caretti, A. Noll, Y.J. Tang, S. Nagaraja, E.M. Gibson, C.W. Mount, J. Polepalli, S.S. Mitra, P.J. Woo, R.C. Malenka, H. Vogel, M. Bredel, P. Mallick, M. Monje, Neuronal activity promotes glioma growth through neuroligin3 secretion, Cell 161 (2015) 803–816. [7] P. Jobling, J. Pundavela, S.M.R. Oliveira, S. Roselli, M.M. Walker, H. Hondermarck, Nerve-cancer cell cross-talk: a novel promoter of tumor progression, Cancer Res. 75 (2015) 1777–1781. [8] D.F. Quail, J.A. Joyce, Microenvironmental regulation of tumor progression and metastasis, Nat. Med. 19 (2013) 1423–1437. [9] A.A. Bapat, G. Hostetter, D.D. Von Hoff, H. Han, Perineural invasion and associated pain in pancreatic cancer, Nat. Rev. Cancer 11 (2011) 695–707. [10] I.E. Demir, G.O. Ceyhan, F. Liebl, J.G. D'Haese, M. Maak, H. Friess, Neural invasion in pancreatic cancer: the past, present and future, Cancers 2 (2010) 1513–1527. [11] T. Kiba, Relationships between the autonomic nervous system and the pancreas including regulation of regeneration and apoptosis — recent developments, Pancreas 29 (2004) E51–E58. [12] I. Makino, H. Kitagawa, T. Ohta, H. Nakagawara, H. Tajima, I. Ohnishi, H. Takamura, T. Tani, M. Kayahara, Nerve plexus invasion in pancreatic cancer: spread patterns on histopathologic and embryological analyses, Pancreas 37 (2008) 358–365. [13] G. Burnstock, Neurotransmitters and trophic factors in the autonomic nervous-system, J. Physiol. Lond. 313 (1981) 1–35. [14] L.S. Satin, T.A. Kinard, Neurotransmitters and their receptors in the islets of Langerhans of the pancreas — what messages do acetylcholine, glutamate, and GABA transmit, Endocrine 8 (1998) 213–223. [15] E.R. Spindel, Muscarinic receptor agonists and antagonists: effects on cancer, Handb. Exp. Pharmacol. (2012) 451–468.

141

[16] N. Shah, S. Khurana, K. Cheng, J.P. Raufman, Muscarinic receptors and ligands in cancer, Am. J. Physiol. Cell Physiol. 296 (2009) C221–C232. [17] M.S. Ackerman, W.R. Roeske, R.J. Heck, M. Korc, Identification and characterization of muscarinic receptors in cultured human pancreatic-carcinoma cells, Pancreas 4 (1989) 363–370. [18] J.L. Chien, J.R. Warren, Muscarinic receptor coupling to intracellular calcium release in rat pancreatic acinar carcinoma, Cancer Res. 46 (1986) 5706–5714. [19] R.A. Harvey, P.C. Champe, Lippincott Illustrated Reviews: Pharmacology (4th Edition), Lippincott Williams & Wilkins, 2009. [20] O.C. Farokhzad, R. Langer, Impact of nanotechnology on drug delivery, ACS Nano 3 (2009) 16–20. [21] Y. Tian, H.M. Wang, Y. Liu, L.N. Mao, W.W. Chen, Z.N. Zhu, W.W. Liu, W.F. Zheng, Y.Y. Zhao, D.L. Kong, Z.M. Yang, W. Zhang, Y.M. Shao, X.Y. Jiang, A peptide-based nanofibrous hydrogel as a promising DNA Nanovector for optimizing the efficacy of HIV Vaccine, Nano Lett. 14 (2014) 1439–1445. [22] K. Fan, C. Cao, Y. Pan, D. Lu, D. Yang, J. Feng, L. Song, M. Liang, X. Yan, Magnetoferritin nanoparticles for targeting and visualizing tumour tissues, Nat. Nanotechnol. 7 (2012) 459–464. [23] M. Liang, K. Fan, M. Zhou, D. Duan, J. Zheng, D. Yang, J. Feng, X. Yan, H-ferritinnanocaged doxorubicin nanoparticles specifically target and kill tumors with a single-dose injection, Proc. Natl. Acad. Sci. U. S. A. 111 (2014) 14900–14905. [24] C.Q. Cao, X.X. Wang, Y. Cai, L. Sun, L.X. Tian, H. Wu, X.Q. He, H. Lei, W.F. Liu, G.J. Chen, R.X. Zhu, Y.X. Pan, Targeted in vivo imaging of microscopic tumors with ferritinbased nanoprobes across biological barriers, Adv. Mater. 26 (2014) 2566–2571. [25] E.C. Theil, Ferritin: structure, gene regulation, and cellular function in animals, plants, and microorganisms, Annu. Rev. Biochem. 56 (1987) 289–315. [26] M. Uchida, M.L. Flenniken, M. Allen, D.A. Willits, B.E. Crowley, S. Brumfield, A.F. Willis, L. Jackiw, M. Jutila, M.J. Young, T. Douglas, Targeting of cancer cells with ferrimagnetic ferritin cage nanoparticles, J. Am. Chem. Soc. 128 (2006) 16626–16633. [27] X. Lin, J. Xie, G. Niu, F. Zhang, H.K. Gao, M. Yang, Q.M. Quan, M.A. Aronova, G.F. Zhang, S. Lee, R. Leaprnan, X.Y. Chen, Chimeric ferritin nanocages for multiple function loading and multimodal imaging, Nano Lett. 11 (2011) 814–819. [28] Z.P. Zhen, W. Tang, H.M. Chen, X. Lin, T. Todd, G. Wang, T. Cowger, X.Y. Chen, J. Xie, RGD-modified apoferritin nanoparticles for efficient drug delivery to tumors, ACS Nano 7 (2013) 4830–4837. [29] T.R. Daniels, E. Bernabeu, J.A. Rodriguez, S. Patel, M. Kozman, D.A. Chiappetta, E. Holler, J.Y. Ljubimova, G. Helguera, M.L. Penichet, The transferrin receptor and the targeted delivery of therapeutic agents against cancer, Biochim. Biophys. Acta Gen. Subj. 1820 (2012) 291–317. [30] L. Li, C.J. Fang, J.C. Ryan, E.C. Niemi, J.A. Lebron, P.J. Bjorkman, H. Arase, F.M. Torti, S.V. Torti, M.C. Nakamura, W.E. Seaman, Binding and uptake of H-ferritin are mediated by human transferrin receptor-1, Proc. Natl. Acad. Sci. U. S. A. 107 (2010) 3505–3510. [31] E.N. Marieb, Essentials of Human Anatomy and Physiology, 10th ed., 2012. [32] E.D.B. Clark, Refolding of recombinant proteins, Curr. Opin. Biotechnol. 9 (1998) 157–163. [33] I.F. Tannock, D. Rotin, Acid pH in tumors and its potential for therapeutic exploitation, Cancer Res. 49 (1989) 4373–4384. [34] L.E. Gerweck, K. Seetharaman, Cellular pH gradient in tumor versus normal tissue: potential exploitation for the treatment of cancer, Cancer Res. 56 (1996) 1194–1198. [35] J.L. Zhang, W. Jin, X.Q. Wang, J.C. Wang, X.A. Zhang, Q.A. Zhang, A novel octreotide modified lipid vesicle improved the anticancer efficacy of doxorubicin in somatostatin receptor 2 positive tumor models, Mol. Pharm. 7 (2010) 1159–1168. [36] H. Maeda, J. Wu, T. Sawa, Y. Matsumura, K. Hori, Tumor vascular permeability and the EPR effect in macromolecular therapeutics: a review, J. Control. Release 65 (2000) 271–284. [37] M.J. Carden, J.Q. Trojanowski, W.W. Schlaepfer, V.M.Y. Lee, 2-stage expression of neurofilament polypeptides during rat neurogenesis with early establishment of adult phosphorylation patterns, J. Neurosci. 7 (1987) 3489–3504. [38] S. Elmasry, S.A.H. Khalil, Adsorption of atropine and hyoscine on magnesium trisilicate, J. Pharm. Pharmacol. 26 (1974) 243–248. [39] G.M. Yan, S.Z. Lin, R.P. Irwin, S.M. Paul, Activation of muscarinic cholinergic receptors blocks apoptosis of cultured cerebellar granule neurons, Mol. Pharmacol. 47 (1995) 248–257. [40] S.C. Yang, H.X. Ge, Y. Hu, X.Q. Jiang, C.Z. Yang, Doxorubicin-loaded poly(butylcyanoacrylate) nanoparticles produced by emulsifier-free emulsion polymerization, J. Appl. Polym. Sci. 78 (2000) 517–526. [41] E. Ryschich, G. Huszty, H.P. Knaebel, M. Hartel, M.W. Büchler, J. Schmidt, Transferrin receptor is a marker of malignant phenotype in human pancreatic cancer and in neuroendocrine carcinoma of the pancreas, Eur. J. Cancer 40 (2004) 1418–1422. [42] P. Song, H.S. Sekhon, A. Lu, J. Arredondo, D. Sauer, C. Gravett, G.P. Mark, S.A. Grando, E.R. Spindel, M3 muscarinic receptor antagonists inhibit small cell lung carcinoma growth and mitogen-activated protein kinase phosphorylation induced by acetylcholine secretion, Cancer Res. 67 (2007) 3936–3944. [43] S.J. Miknyoczki, D. Lang, L.Y. Huang, A.J.P. Klein-Szanto, C.A. Dionne, B.A. Ruggeri, Neurotrophins and trk receptors in human pancreatic ductal adenocarcinoma: expression patterns and effects on in vitro invasive behavior, Int. J. Cancer 81 (1999) 417–427. [44] Z.W. Zhu, H. Friess, F.F. diMola, A. Zimmermann, H.U. Graber, M. Korc, M.W. Buchler, Nerve growth factor expression correlates with perineural invasion and pain in human pancreatic cancer, J. Clin. Oncol. 17 (1999) 2419–2428. [45] M.B. Schneider, J. Standop, A. Ulrich, U. Wittel, H. Friess, A. Andren-Sandberg, P.M. Pour, Expression of nerve growth factors in pancreatic neural tissue and pancreatic cancer, J. Histochem. Cytochem. 49 (2001) 1205–1210.

142


[46] Z.W. Zhu, H. Friess, L. Wang, T. Bogardus, M. Korc, J. Kleeff, M.W. Buchler, Nerve growth factor exerts differential effects on the growth of human pancreatic cancer cells, Clin. Cancer Res. 7 (2001) 105–112. [47] R.E. Stopczynski, D.P. Normolle, D.J. Hartman, H.Q. Ying, J.J. DeBerry, K. Bielefeldt, A.D. Rhim, R.A. DePinho, K.M. Albers, B.M. Davis, Neuroplastic changes occur early in the development of pancreatic ductal adenocarcinoma, Cancer Res. 74 (2014) 1718–1727. [48] Z.W. Zhu, J. Kleeff, H. Kayed, L. Wang, M. Korc, M.W. Buchler, H. Friess, Nerve growth factor and enhancement of proliferation, invasion, and tumorigenicity of pancreatic cancer cells, Mol. Carcinog. 35 (2002) 138–147. [49] P.X. Liu, H.L. Cheng, T.M. Roberts, J.J. Zhao, Targeting the phosphoinositide 3-kinase pathway in cancer, Nat. Rev. Drug Discov. 8 (2009) 627–644.

[50] M. Mancino, E. Ametller, P. Gascon, V. Almendro, The neuronal influence on tumor progression, BBA-Rev Cancer 1816 (2011) 105–118. [51] G.O. Ceyhan, I.E. Demir, B. Altintas, U. Rauch, G. Thiel, M.W. Muller, N.A. Giese, H. Friess, K.H. Schafer, Neural invasion in pancreatic cancer: a mutual tropism between neurons and cancer cells, Biochem. Biophys. Res. Commun. 374 (2008) 442–447. [52] D. Hanahan, R.A. Weinberg, Hallmarks of cancer: the next generation, Cell 144 (2011) 646–674. [53] A.C. Kruse, B.K. Kobilka, D. Gautam, P.M. Sexton, A. Christopoulos, J. Wess, Muscarinic acetylcholine receptors: novel opportunities for drug development, Nat. Rev. Drug Discov. 13 (2014) 549–560.