Efficient Targeting Drug Delivery System for Lewis Lung Carcinoma

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An Efficient Targeting Drug Delivery System for Lewis Lung Carcinoma, Leading Histomorphological Abnormalities Restoration, Physiological and Psychological Statuses Improvement, and Metastasis Inhibition Ye Yang, Hanxu Cai, Xiuyan Yuan, Huihui Xu, Yingying Hu, Xue Rui, Jingjing Wu, Jing Chen, Jing Li, Xiangdong Gao, and Dengke Yin Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.8b00161 • Publication Date (Web): 11 Apr 2018 Downloaded from http://pubs.acs.org on April 13, 2018

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Molecular Pharmaceutics

An Efficient Targeting Drug Delivery System for Lewis Lung Carcinoma, Leading Histomorphological Abnormalities Restoration, Physiological and Psychological Statuses Improvement, and Metastasis Inhibition Ye Yang†,‡,§, Hanxu Cai†, Xiuyan Yuan†, Huihui Xu†, Yingying Hu†, Xue Rui†, Jingjing Wu†, Jing Chen†, Jing Li⊥, Xiangdong Gao*,⊥, and Dengke Yin∗,†,‡,§ †

School of Pharmacy, Anhui University of Chinese Medicine, Hefei 230012, P.R.

China ‡

Institute of Pharmaceutics, Anhui Academy of Chinese Medicine, Hefei, 230012,

PR China §

Anhui Province Key Laboratory of R&D of Chinese Medicine, Hefei 230012, P.R.

China ⊥ Jiangsu

Key Laboratory of Druggability of Biopharmaceuticals, China

Pharmaceutical University, Nanjing 210009, P.R. China

Abstract Lung cancer is a kind of malignant tumour with high morbidity and metastasis tendency. Gambogic acid (GA) has demonstrated and significant antitumor activity in vitro, but its poor water-solubility and adverse effects restrict its application in vivo and in clinic. In this study, a passive-targeting GA delivery system was prepared for orthotopic Lewis lung carcinoma model mice. Besides of the 7 µm around size distribution, slow and steady in vitro drug release in a week, high targeting effect to lung, effective restoration of histomorphological abnormalities in lung, maintaining on bodyweight, and prolongation on survival time, the excellent improvements of the GA-loaded particles on physiological and psychological statuses, and obvious inhibition on tumour metastasis to liver had also been observed, through the measurements of porsolt forced swim, hypoxic tolerance time, ultrastructure of pulmonary capillary, pulmonary vascular permeability, and hepatic histological ∗

Corresponding Author: Address: 1 Qianjiang Road, Xinzhan District, Hefei 230012; Tel: +86 551

68129125; Fax: +86 551 68129125; E-mail address: [email protected] (D. Yin); [email protected] (X. Gao)

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change. These results suggest that this GA-loaded particle may be an ideal approach to achieve satisfactory therapeutic function on lung cancer.

KEYWORDS: targeting drug delivery system, orthotopic Lewis lung carcinoma, gambogic acid (GA), physiological and psychological status, tumour metastasis

Introduction Gambogic acid (GA, C38H44O8, Figure 1a) is the main active component isolated from the dry gum-resin of Garcinia hanburyi and possesses many kinds of pharmacological activity,1 the most prominent of which is anti-tumour activity.2 A lot of reports show that there are several acting mechanisms on the anti-tumour effects of GA, such as inducing cell apoptosis and differentiation, inhibiting cell proliferation and transference, reversing multidrug-resistant, regulating immunity, and so on.2-3 Lung cancer is one of the leading causes of cancer-related death worldwide. As tumour angiogenesis is an important dependency factor of the proliferation and metastasis of lung cancer, anti-angiogenesis is a widely accepted therapeutic target of lung cancer.4-5 The anti-angiogenesis effect of GA has been fully proved, however, most experiments were carried out at the cellular level6-7 or through local application.8-9 It is largely on account of the serious vascular stimulation, poor aqueous solubility (~ 10 µg/mL) and short half-life of GA.10 Structural modification10 and drug delivery system11-13 are the common methods to solve these problems. Compared to the complexity of structural modification, a drug delivery system with appropriate carrier material and designed ultrastructure can not only successfully solve the problems above but also achieve targeted and controlled drug delivery. In our earlier study of GA, a series of GA-loaded poly-DL-lactide (PDLLA) particles with range in size from tens of nanometers to several micrometers were prepared and exhibited targeting effects to different tissues, and the most effective of which was the liver-targeting particle with size of 200 nm.14 Although the GA-loaded PDLLA particles with different size ranges exhibited targeting effects to different tissues/organs, which were corresponded with the laws of passive-targeting,15 the drug distribution in liver usually dominate. The greater mass of liver and the lipophilic surface of GA-loaded PDLLA particle may be the probable reason for this phenomenon. Besides of size distribution, the material properties of the drug carriers are the other important influence factors and cannot be ignored in tissue targeting. ACS Paragon Plus Environment

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Hydrophilic and positively charged surface have been reported to induce prolonged circulation and enhanced endocytosis of drug delivery systems.16-18 Thus, in the present study, a hydrophilic, positively charged, and lung-targeting GA delivery system was designed and prepared. After the detection of the conventional parameters of drug delivery system, such as surface morphology, size distribution, drug loading amount, encapsulated efficiency, and in vitro drug release profile, the targeting efficiency to lung and anti-tumour effect to orthotopic Lewis lung carcinoma (LLC) model mice were also investigated, with comparison to GA solution. The antitumour effect of this GA delivery system was evaluated not only by the histological change of lung tissue, physical state, and survival time, but also by the lung function, psychological status, and tumour metastasis to the liver.

Materials and Methods Materials. Gambogic acid (GA; purity ≥ 99.0%) was donated from professor He in our University. Poly(lactic-co-glycolic acid (PLGA, DL-LA/GA = 50 ∶ 50, Mw = 35 kDa) was purchased from Jinan Daigang Biomaterial Co., Ltd (Shandong, China). High molecular weight chitosan (CS; Mw = 500 kDa, deacetylation degree = 85%) was obtained from Chengdu Kelong Reagent Co. (Chengdu, China). The fluorescent marker of 1,3,6-pyrenetrisulfonicacid, 8-amino-, sodium salt (1:3) (APTS) was purchased from Aladdin Reagent Database Inc. (Shanghai, China). Rabbit anti-mouse antibodies specific to CD31, biotinylated secondary antibody, streptavidinhorseradish peroxidase (HRP) and 3,3’-diaminobenzidine (DAB) were purchased from Biosynthesis Biotechnology Co., Ltd (Beijing, China). All other chemicals and solvents were of analytical grade or better and were purchased from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China).

Animal. The inbred male C57BL/6 mice (weight = 18-20 g, age = 4-6 weeks) were supplied by the SLRC Laboratory Animal Company (Shanghai, China), allowed to acclimatization for 1 week before experimentation, fed with a standard diet, and allowed water ad libitum.

Ethics statement. The animal experimental protocol was reviewed and approved by the Institutional Ethics Committee of the Anhui University of Chinese Medicine. All the experiments were carried out in accordance with the approved guidelines. ACS Paragon Plus Environment


Cells and cell culture. LL/2 cells were preserved by our lad and used to establish the model of Lewis Lung Carcinoma (LLC) mice. The cells were cultured in Dulbecco's Modified Eagle's Medium (Gibco BRI, Rockville, MD) with 10% heat-inactivated fetal calf serum (Gibco BRI, Grand Island, NY) and placed in a 75 cm2 cell-culture flask at 37 °C with 5% CO2 in the air. GA-loaded particle preparation. One hundred milligrams of chitosan was dissolved in 10 mL of 4% acetic acid aqueous solution (w/v, pH 4.0). A chloroform solution of GA and PLGA, with a mass-volume concentration of 4 and 100 mg/mL, was dropped into the chitosan solution with volume rate of 1 ∶ 10, under ultra-sonication in an ice bath (VC 505, Sonics & Materials Inc., Newtown, CT). The oil/water emulsion was electrosprayed by an electrospray apparatus, which was equipped with a high-voltage statitron (Tianjing High Voltage Power Supply Co., Tianjing, China), a microinject pump (Zhejiang University Medical Instrument Co., Hangzhou, China) and a 2 mL syringe, with technological parameters of 1.0 mL/min flow rate, 1.19 mm capillary inner diameter, 13 kV applied voltage, and 15 cm receiving distance to aqueous solution of ammonia (pH 9.0). The obtained particle suspension was quickly stirred to remove the residual solvent and then adjusted pH value to neutral. The particles were named as CS/PLGA-GA. APTS-labelled GA was synthesized in our lab according to the reported method.19 Then the APTS-GA-loaded particles were prepared according to the above method and parameters and named as CS/PLGA-A-GA. Blank particles without GA content were also prepared and named as CS/PLGA. All the particles were collected after freeze drying.

Particle characterization. The size distribution and surface potential of CS/PLGAGA and CS/PLGA-A-GA were detected using a dynamic light scattering (DLS) (Malvern Nano-2S90, Malvern, U.K.) at 25 °C after ultrasonic dispersion. The morphology of CS/PLGA-GA and CS/PLGA-A-GA were observed after mounting on metal stubs, sputter coating with gold, and detecting at an accelerating voltage of 15 kV, using a scanning electron microscope (SEM; FEI Quanta 200, The Netherlands). Drug loading amount and encapsulated efficiency of GA into particles were determined by the high-performance liquid chromatography (HPLC). In brief, 20 mg of CS/PLGA-GA was dissolved in 2.0 mL of 4% acetic acid, extracted by 1.0 mL of ACS Paragon Plus Environment

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chloroform, centrifuged at 20,627 × g for 10 min using a Microfuge 20R high-speed centrifuge (Beckman Coulter, Inc., CA, USA) and filtered by 0.22 µm microfiltration membrane. The GA content was detected by HPLC system, with Waters 2695 (Milford, MA) as the separations module, C18 Hypersil BDS (4.6 mm × 250 mm × 5 µm, Hypersil, Runcorn, U.K.) as the analytical column, mixed solution of methanol and 0.1% phosphoric acid (95:5, v/v) as the mobile phase at a flow rate of 1.0 mL/min, and Waters 2487 (Milford, MA) as the dual absorbance ultraviolet detector at a detected wavelength of 360 nm. The concentration of GA was calculated using a standard curve prepared from a series of GA solution with known concentrations and from the corresponding peak area. The GA loading amount and encapsulated efficiency were calibrated by the extraction efficiency of chloroform on GA.

In vitro GA release. One hundred milligrams of CS/PLGA-GA were incubated in 10.0 mL of phosphate buffer (PBS, pH 7.2-7.4) with 0.5% (w/v) of sodium dodecyl sulphate (SDS)20 and 0.02% (w/v) sodium azide. The thermostatic shaking water bath (Taichang Medical Apparatus Co., Jiangsu, China) with a constant temperature of 37 °C and rotate speed of 100 cycles/min was used for the in vitro drug release. A half milliliter of the release buffer was taken out at the predetermined time points and replaced by fresh buffer. The release profile of GA from particles was obtained according to the detection of GA content in release buffer, by the HPLC method described above. Blank particles of CS/PLGA were used as a control for the GA amount determination.

Tissue targeting and drug distribution. One hundred and eight mice were randomized into 3 groups, respectively tail-vein-injected with CS/PLGA-GA suspension (2.5 mg/kg), GA solution (2.5 mg/kg), and the same volume of normal saline (NS), and named as group GA-P, GA-S, and NS. At the predetermined time points of 5, 10, 15, 30, 60, and 90 min after dosing, 6 mice in each group were sacrificed and tissues from heart, liver, spleen, lung, and kidney were excised, weighed, grinded with liquid nitrogen, deproteinized using perchloric acid, extracted using ethyl acetate, blow-dried using nitrogen at 40 °C in a water bath, redissolved in methanol, and analyzed for GA content using HPLC. The tissue-specific effects of GA solution and CS/PLGA-GA particle were eliminated by respectively adding a ACS Paragon Plus Environment


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series of GA solutions or CS/PLGA-GA suspensions into each type tissue homogenate and preparing GA standard curves for each tissue with the same processes in accordance with the methods described above. Drug-time curves of GA in tissues were obtained and the targeting effects of GA solution and CS/PLGA-GA particle were calculated with indices of relative tissue exposure (re), drug targeting index (DTI), targeting efficiency (Te), and relative targeting efficiency (RTe), according to the following equations:21 re=

(AUCi )p (AUCi )s

DTI= T e=

[GA concentration(t )i × ( weight of tissue)i ]p [GA concentration(t )i × ( weight of tissue)i ]s AUCi × (weight of tissue)i

n

×100%

∑ [AUCi × (weight of tissue)i] i =1

n

(Te)p RTe= = (Te)s

[ AUCi × (weight of tissue)i ]

∑ [AUC × (weight of tissue) ] i

i p

i =1 n

[ AUCi × (weight of tissue)i ]

∑ [AUC × (weight of tissue) ] i

i s

i =1

To observe the drug distribution in different tissues, A-GA solution, CS/PLGA-AGA particle suspension, and normal saline were also tail-vein-injected in accordance with the method described above, and tissues of heart, liver, spleen, lung, and kidney were excised, fixed, paraffin-embedded, sliced up, and observed by fluorescence microscope (Leica DMR HCS, Germany).

Mouse orthotopic Lewis lung carcinoma (LLC) model creation. The LLC mouse model was established as described previously.22 The LL/2 cell suspension was triply rinsed with normal saline, centrifuged at 3,000 r/min for 1 min, suspended with normal saline to a concentration of 1 × 107 tumour cells/mL, and mixed with Matrigel matrix (Corning Costar, Cambridge, MA, USA) at a volume rate of 1 ∶ 1. The C57BL/6 mice were intraperitoneally injected with pentobarbital sodium (10 mg/kg) to anesthesia, fixed in the supine position, disinfected the skin, made a 0.6 cm longitudinal incision in the skin of neck by aseptic eye scissor, blunt separated the paratracheal soft tissue, exposed the trachea, injected with 100 µL of LL/2 cell gel (5 × 105 cells) into the trachea, erected and gently vibrated the body to allow the flow of cell suspension, sutured the incision, and then maintained in the supine position until ACS Paragon Plus Environment

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complete recovery. The mice in the control group underwent the same treatment but were injected with normal saline.

Dosage regimen design. One hundred and twenty LLC model mice were randomly and evenly divided into 4 groups. The three of the LLC model groups were tail-vein injected with CS/PLGA-GA suspension (2.5 mg/kg dose, GA-P group), GA solution (2.5 mg/kg dose, GA-S group), and normal saline (NS group), respectively, while the other group of LLC model mice was given no injection (model group). The normal mice (normal group) were used as controls.

Physical state. The physical state of LLC mice in each experimental group was daily observed and documented, such as hair quality, ingestion, drinking, and body weight.

Hypoxic tolerance time test (HTTT). Seven days after administration, one-fifth of mice in each group were individually placed into a 125 mL reagent bottle with fresh and atmospheric air and 3 gram of soda lime (for the absorption of water and carbon dioxide) and then sealed with a rubber plug. The hypoxic tolerance limit was judged by the appearance of gasping breath and the time period between the beginning of airtightness and the appearance of the first gasping was termed “original tolerance time” (To). The “standard tolerance time” (Ts) was calculated according to the following equations:23-25 T s=

To To ×100= ×100 Ve V 0 - ma × 0.94

Ve: effective bottle volume; Vo: original bottle volume; ma: body weight of the mouse.

Porsolt forced swim test (PFST). Seven days after administration, one-fifth of mice in each group were tied with 7% body weight of plasticine on the tail-end and individually placed in a non-escapable cylinder (30 cm height × 15 cm diameter), half-filled with water at 25 ± 1 °C. The time period between the beginning of swimming and the appearance of immobility was manually recorded. The immobile state was considered when only slight movement could be observed, in order to keep head above water.26-28



Histological examinations. Seven days after administration, one-fifth of mice in each group were sacrificed and the left pulmonary lobes and the hepatic lobes were excised. About 1 cm3 of tissue were excised, fixed, paraffin-embedded, sectioned, and stained by hematoxylin-eosin (HE) to observe the tissue histomorphology. In brief, the sections were gradually immersed in formaldehyde (10%, v/v), washed with distilled water, stained nuclei with hematoxylin, rinsed in running tap water, differentiated with acid alcohol (0.3%, v/v), rinsed in running tap water, stained with eosin for 2 min, dehydrated, cleared, mounted, and then observed using a light microscope (Nikon Eclipse E400, Japan). For electron microscopy, small pieces of the lung tissues were fixed with 2.5% glutaraldehyde, postfixed with 1% osmium tetroxide, rinsed and dehydrated in graded ethanol, processed by propylene oxide, and embedded in epoxy resin. Ultrathin sections (0.07 µm) were cut using a diamond knife by ultramicrotome (LKB-NOVA, Sweden) and then stained with uranyl acetate and lead citrate, and investigated with a JEM-1230 transmission electron microscope (TEM, JEOL, Tokyo, Japan).

Immunohistochemistry (IHC) staining. The sections of tumour tissues were IHCstained of CD31 to evaluate the microvessels. In brief, the sections were gradually immersed in H2O2 (3%, v/v) and citrate buffer solution (10 mM, pH 6.0), heated in a microwave oven, washed with PBS (10 mM, pH 7.2), blocked with bovine serum albumin in Tris-buffered saline (5%, w/v), drained excess liquid, incubated with primary antibody at 4 °C overnight, washed with PBS, sequentially incubated with biotinylated secondary antibody for 20 min, streptavidin-HRP for 20 min, and DABH2O2, counterstained with haematoxylin, dehydrated, hyalinised, and mounted. Then, the IHC-stained sections were observed with a light microscope, randomly photographed under the same magnification, and then calculated the pixel values of pale brown coloured positive CD31 per square centimetre, which were defined as relative CD31 level, by the software of Image Tool program (V. 3.0, The University of Texas Health Science Center, San Antonio, TV). To avoid the artificial error, five pictures were randomly photographed in each tissue and the mean values of the six samples in each experimental group were calculated. For the microvessel density (MVD), an endotheliocyte cluster of the pale brown colour of CD31 expression was identified as a blood vessel, regardless of whether the lumen or red blood cells were present, according to the method of Weidner.29


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Pulmonary vascular permeability (PVP) index. Seven days after administration, onefifth of mice in each group were tail-vein injected with 100 µL 5% evans blue (w/v). Thirty minutes after injection, blood samples were obtained via the retinal vein plexus and centrifuged for supernatants. Then the mice were sacrificed, sliced from the neck down to the chest, exposed and tied the left trachea and pulmonary lobe, inserted the puncture needle into the upper trachea, perfused with 0.3 mL of normal saline for 3 times, and collected the perfusates.30 The blood supernatants and perfusates were measured by reading the absorbance at 620 nm. Absorbance ratios of perfusate to blood supernatant were calculated to denote the pulmonary vascular permeability.

Survival time. The last one-fifth of the LLC model mice in each group were fed in a conventional manner until natural death. The survival times in each group were observed.

Statistical analysis. The results were expressed as means ± standard deviation of percentage values of the basal level (n = 6). Statistical significance of differences was examined using one-way analysis of variance followed by LSD post hoc test. A p value of < 0.05 was considered to be statistically significant.

Results Particle characterization. The DLS result (as shown in Figure 1b) indicates that the size distribution of GA-P is 7.39 ± 1.51 µm. SEM image (Figure 1c) shows that the CS/PLGA-GA particles are ellipsoidal shaped with a few of structural defect. The GA amount and encapsulated efficiency in CS/PLGA-GA particles are (3.44 ± 0.18)% and (86.00 ± 5.15) %, respectively.

In vitro GA release. In the in vitro GA release experiment, the sink condition was met. Figure 1d is the GA release profile from particles with accumulated GA release rate as ordinate. The burst release in initial 24 h is lower than 3%, and the subsequently accelerated release phase lasts one week and releases about 90% of the drug.



Tissue targeting and drug distribution. Figure 2a1 and 2a2 respectively show the GA concentrations in tissues versus time, after tail-vein injection of GA solution and GAloaded particles. In the GA-S group (Figure 2a1), GA reaches its maximal concentration (Cmax) in each tissue immediately after the tail-vein injection and drops swiftly over time. Besides the transiently higher GA concentration in liver than the other tissues (p < 0.05) after injection, GA is widely and evenly distributed in all the dominate tissues. In the GA-P group (Figure 2a2), the GA concentration in the lung tissue is significantly and continuously higher than those in other tissues (p < 0.05) since 10 min postdose, which is also the time of peak GA concentration (Tmax). Moreover, the concentrations of GA in the lung tissue in the GA-P group are always much higher than that in the liver in the GA-S group, at the same time points since the Tmax. Figure 2b shows the ratio of the GA-amount in tissues (GA concentrationi × weight of tissueDT) in the GA-P group to the GA-amount in the GA-S group, designated as DTI. Five minutes postdose, the DTI of all the dominant tissues are close to zero. Since 10 min postdose, the DTI of the lung tissue continues at a high level of 3.78 ± 1.10, 6.43 ± 1.40, 3.33 ± 0.63, 2.51 ± 0.39, and 2.56 ± 0.42, indicating up to 6.43times higher GA amount in the lung in the GA-P group compared with the GA-S group. Table 1 summarizes the areas under the concentration-time curve (AUC) and the distribution ratios (Te) of GA in all the dominate tissues after administrations, and the AUC ratios (re) and the Te ratios (RTe) between the GA-P group and GA-S group. As shown in Table 1, the re and RTe of the lung in the GA-P group is 2.88 ± 0.18 and 3.76 ± 0.20, indicating 2.88-times and 3.76-times higher GA AUC and GA distribution ratio than those of GA-S group. The lower than 1 values of re and RTe in the heart, liver, and spleen indicate the remarkable reduction of GA distribution in these tissues. To observe the GA distribution in different tissues, APTS-GA-loaded particle, which exhibits the same characterization with CS/PLGA-GA, was administrated to the model mice, as well as APTS-GA solution. Figure 2c shows the fluorescent microscope images of tissues at 10 min postdose. In A-GA solution group, the yellow-green fluorescence of APTS-labelled GA can be observed in all tissues and is strongest in the liver. In CS/PLGA-A-GA group, the fluorescence intensity in the lung tissue is much enhanced, while the fluorescence intensities in other tissues are weakened.


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Table 1. Mean values of GA AUC (µg × min/g), Te (%), re, and RTe in tissues after tail-vein injection of GA solution or GA-loaded particles, at a dose equivalent to GA 2.5 mg/kga. Groups GA-S AUCi Heart Liver

a

GA-Ps Te (%)

Te (%)

RTe

AUCi

re

2.25 ± 0.47

32.03 ± 10.12

0.62 ± 0.08

2.00 ± 1.25

0.88 ± 0.17

137.23 ± 23.27 b 79.73 ± 6.76

79.70 ± 14.43

0.58 ± 0.10

64.24 ± 7.75

0.80 ± 0.15

50.28 ± 11.81

0.66 ± 0.14

1.77 ± 1.12

0.96 ± 0.16

50.20 ± 16.06

Spleen

72.38 ± 12.42

1.83 ± 0.82

Lung

74.81 ± 13.28

5.20 ± 1.01

Kidney

75.12 ± 15.42

10.98 ± 1.64 b

202.71 ± 21.16b, * 2.88 ± 0.18b 61.35 ± 15.50

0.82 ± 0.02

*

Data are presented as the mean ± SD (n = 6). p < 0.05 vs other tissues. p < 0.05 vs GA-S group.


19.55 ± 2.83 * 3.76 ± 0.20b 12.44 ± 1.20

1.13 ± 0.18


LLC model creation. After LL/2 cells injection, the mice are monitored continuously to ensure the best environment possible for recovery. The pilot experiments show that nearly 100% of the modelling mice show progressive emaciation, declining food intake, irregular hair coat, and poor spirit. The histological examination exhibits glandular configurations, which can be identified as successful LLC mice model.22

Lung function. Figure 3a summarizes the Ts of LLC model mice after various administrations. The modelling of lung cancer induces significantly decrease of Ts (p < 0.05). Although both the administration of GA solution and the GA-loaded particle can significantly increase the Ts of LLC mice (p < 0.05), the drug-loading particle exhibits much better effect (p < 0.05). Administration of normal saline exhibits no effect on Ts.

Bodyweight. Figure 3b summarizes the bodyweight of LLC model mice in each experimental group. It shows that the average bodyweight of normal health mice increases slowly and steadily with time elapsed, however, the weight of LLC model mice continuously loses. No maintaining effect on bodyweight can be observed in group NS; and the groups of GA-S and GA-P exhibit similarly and significantly maintaining effect on the bodyweight of LLC model mice and exhibit significantly higher average bodyweight than the mice in model group and NS group (p < 0.05).

Behavioural feature. Figure 3c summarizes the PFST of LLC model mice after various administrations. The modelling of lung cancer induces significantly decrease of swimming time (p < 0.05). Both the group of GA-S and GA-P exhibit a significant improvement of the swimming time, but the GA-loaded particle induces much better effect (p < 0.05). The group of NS exhibits similar swimming time with the model group.

Morphologic changes of lung tissue. The micrograph of lung tissue in normal mice exhibits typical bronchus architecture with columnar epithelial cells, basophilic goblet cells, and massive pulmonary alveolus, consisting of large and flat alveolar type I cells, cuboidal alveolar type II cells with ovoid nuclei, and connective tissue surrounded fibroblasts (not shown in this article). Figure 4a shows the pulmonary histomorphology of LLC model mice in each experimental group at the seventh day


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after administration, using HE staining. In LLC model group (Figure 4a), glandular configurations with squamous cell hyperplasia, pleomorphic cells and hyperchromatic nuclei (dashed line bounded area), alveolar type II cell hyperplasia in alveolar septa (defined as AS), and atrophic alveoli (defined as A) can be observed. The lung tissue in NS group (Figure 4b) exhibits almost the same morphologic characteristics as the LLC model mice. The pulmonary histomorphology in GA-S group (Figure 4c) exhibits simple cuboidal or columnar epithelium (black arrow) covered terminal bronchioles (defined as TB), alveolar septa (defined as AS) with type 1 and type 2 pneumocytes, alveolus (defined as A), and close-packed tumour cells with thin cytoplasm and hyperchromatic nuclei (yellow arrow), without any glandular configuration. The pulmonary histomorphology in GA-P group (Figure 4d) exhibits similar morphologic characteristics with the GA-S group, but rare tumour cell.

Tumour angiogenesis. To evaluate the tumour angiogenesis in various administrations, CD31 expression in lung tissues were detected by IHC. Figure 5a shows the representative photographs of lung tissues (Figure 5a1) in each experimental group and the image data analysis of relative CD31 level (Figure 5a2) and MVD (Figure 5a3). In LLC model mice, the photo of lung tissue exhibits massive CD31 positive expression. The GA administration group, especially the GA-P group, exhibits much lower CD31 expression than that in model group (p < 0.05), meanwhile, the NS group exhibits similar CD31 positive level with the LLC model group. The data analysis of relative CD31 level in photos also exhibits the corresponding results with microscopy. The MVD analysis exhibits that the administration of GA can significantly decrease the MVD in lung tissues in LLC model mice (p < 0.05), especially the dosage form of particle delivery system, while the normal saline has no effect on it.

Tumour metastasis. As shown in Figure 5b, the ultrastructure of lung tissue in LLC model mice shows multiple gaps (dashed line bounded area) in the wall of the tinny capillary and blood platelets enriched near the gaps. Figure 5c summarizes the absorbance ratios of bronchoalveolar perfusates to serum, which is identified as the index of PVP. The PVP of the model group is significantly higher than that of normal mice (p < 0.05). The administration of GA-loaded particle can significantly decrease



the PVP level in LLC mice (p < 0.05), meanwhile, the normal saline exhibits no effect (p > 0.05). Figure 5d shows the representative photographs of liver tissues in each experimental group. In LLC model mice, the photo of liver tissue exhibits heteromorphic and irregularly arranged hepatic cells, basophilic cytoplasts, pleomorphic, hyperchromatic, and vesicular nuclei, and increased nucleo-cytoplasmic ratio. In the NS group, the liver tissue exhibits almost the same morphologic characteristics as the LLC model mice. In the GA-S group, the liver histomorphology exhibits some vesicular and heteromorphic nuclei and globular bodies in the cytoplasm. Meanwhile, the liver histomorphology of LLC mice in GA-P group exhibits uniformly arranged polygonous hepatic cells with a rounded nucleus in the center, which is almost the same as the normal liver.

Survival time. Figure 3d summarizes the survival times of LLC mice in each experiment group. It shows that the survival times of mice in the LLC model group, NS group, GA-S group, and GA-P group are 26.7 ± 5.3, 25.3 ± 4.3, 33.2 ± 6.4, and 48.2 ± 9.3 days, respectively. The survival time of LLC mice in GA-P group are much longer than the other groups (p < 0.05), even than the GA-S group (p < 0.05).

Discussion GA has demonstrated antitumor activity in vitro, but its poor water-solubility and adverse effects restrict its application in vivo and in clinic. Drug delivery system is identified to be a promising strategy to achieve drug encapsulation and tissue targeting. In our previous study, GA-loaded PLGA particles31 and GA-grafted heparin micelles14 with the size distribution of 200-300 nm were prepared and exhibited excellent performances on liver targeting and anti-hepatoma effects. Besides of the size distribution, the material properties of drug carriers are also the important influence factors and cannot be ignored in tissue targeting. For example, hydrophilic and positively charged surface provide prolonged circulation and enhanced endocytosis of drug delivery systems.16-18 In the present study, chitosan-coated PLGA/GA particle was designed and prepared for pulmonary tumour therapy. The morphology, size distribution, drug loading, encapsulation efficiency, in vitro GA release profile, tissue targeting, and anti-tumour effects were investigated. The dosage


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was according to the previous studies of GA delivery system and the pilot experiments.31 The CS/PLGA-GA particle exhibits the features of uniform particle size and high drug encapsulated efficiency. Moreover, the in vitro release profile shows an extremely low release amount in initial 24 h, which was even lower than 3%, and a gradually release in following 6 days, with total release amount about 90%, as shown in Figure 1d. Surface drug locating or surrounding is an accepted reason for initial burst release in drug delivery system.32 In the present study, the CS/PLGA-GA particle is obtained from the electrospray of O/W emulsion. We presume that the poor aqueous solubility (∼10 µg/mL) of GA restricts its diffusion from the inner oil phase to outer aqueous phase and leads the low GA amount on/near the particle surface. Besides of the above physicochemical properties, excellent pulmonary targeting effect is the most important feature of the CS/PLGA-GA particle. Targeting drug delivery to diseased tissue/organ is one of the most effective strategies for increasing therapeutic effect and decreasing side effect of drugs, and size control is the most common and simplest method. It is widely accepted that the particles above 7 µm size can be trapped by the capillary exchange beds in the lung, through mechanical filtration, and then be taken into lung tissue and alveoli.15 The charge response between particles and cells is another important basis for the cellular uptake.33 Research of Tang et al.34 showed that only positively charged particles could be ingested by the cells. Thus, in the present study, the lung targeting particle with chitosan coating and the composite core of PLGA and GA is designed and prepared. Compared with general synthetic macromolecule materials for drug carrier, the coating material of chitosan can provide not only hydrophilic but also positively charged particle surface. Experiments of tissue targeting and drug distribution show that the DTI and RTe of chitosan-coated particle CS/PLGA-GA are 6.43- and 3.76times higher than those of GA solution. In our previous study, the PLGA/GA particle with the same size distribution but without coating material of chitosan just exhibited 3.88-times higher DTI and 2.44-times higher RTe than those of GA solution.31 The improved lung targeting in the present study is probably because of the stronger interaction between positively charged CS/PLGA-GA particle surface and negatively charged cell surface, while the PLGA material is negatively charged. However, the interaction between particle and cell is complex and has not been fully studied. The positive charged may led non-specific cell adhesion,35 which is an adverse factor to ACS Paragon Plus Environment


the lung targeting. The non-negligible drug distribution in the other main tissues may because of this. To obtain visual evidence of the targeting drug delivery, the GA was labelled with APTS and entrapped with the same parameters. The fluorescent images of APTS-GA in tissues (Figure 2c) were corresponding to the data of drug distribution. In the present study, the anti-tumour effects of the lung-targeting GA-loaded particle are evaluated on the LLC model mice and compared with GA solution. As the uncertainty of alpha fetal protein and carcinoembryonic antigen on tumour diagnosis and therapy, histological change, lung function, physical state, and behavioural feature are monitored to evaluate the therapeutic effects. The reversion of alveolar structure abnormalities is indeed the strong proof of anti-tumour effects. Above this, improvement of lung function and psychological states, and inhibition of tumour metastasis should be the more advanced requirements for the patients with tumour. HTTT is a conventional measurement to assess the lung function.24-25 The significant increase of Ts than model mice and the approximate value with normal mice indicates the restoration of lung function. Mood problems of anxiety and depression are commonly occurred in the lung cancer patients, inducing negative impact on both the quality of life and the survival time of patients.[36-38] PFST is an important index of active versus passive coping behaviour and always be used to evaluate the depressive state.27-28 The lower PFST of tumour model mice than normal mice indicates the lower desire to live. Thus, the PFST can be regarded as another reflection of therapeutic effect of the GA delivery system. This negatively psychological state was significantly improved by the administration of CS/PLGA-GA. Bodyweight, hair, feeding habits, and survival time are the conventional indicators for the physical state of tumour animal models. The better maintenance of bodyweight and the prolongation of survival time in CS/PLGA-GA group can be regarded as the improvement of life quality. Lung cancer is one of the malignant tumours with high morbidity and metastasis tendency. Besides of high vascularization level, the increase of vascular permeability in tumour microenvironment, which leads the infiltration of tumour cells, has also been confirmed as the beginning step of metastases.[39-41] Thus, the CD31 expression and the PVP index in lung were used to evaluate the metastases tendency in the present study. The high levels of CD31 expression and PVP in lung tissues indicate the high invasion of lung cancer. The much stronger down-regulation of CD31 expression, MVD, and PVP in GA-P group indicates a risk reduction of tumour


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metastasis. About 38~44% of the lung cancer cases will lead hepatic metastases.42 Compared with the metastasis to other tissues, the hepatic metastasis exhibits poor response to chemotherapy and shout lifetime, this is the important factor of the poor favourable of lung tumour.43 In the present study, the liver histomorphologic features of liver tissues in each experimental group indicate the anti-metastasis effect of GA-P to the liver.

Conclusions A GA delivery system of CS/PLGA-GA particle was prepared in the present study and exhibited uniform size of around 7 µm, hydrophilic shell of chitosan, slow and steady in vitro drug release for about 7 days, 3.76-times RTe to lung than GA solution, obvious restoration of histomorphological abnormalities in lung tissue, anti-hypoxia ability of lung function, depressed emotional state, and poor physical state in LLC model mice, and reduced risk of metastasis to other tissues. In summary, this GAloaded particle may be an ideal approach to achieve satisfactory therapeutic function on lung cancer.

Acknowledgments This project was supported by the Open Project of Jiangsu Key Laboratory of Druggability of Biopharmaceuticals (JKLDBKF201704), the National Natural Science Foundation of China (81303239), and the Specialized Research Fund for the Traditional Chinese Medicine Clinical Research Center of China (JDZX2015128).

Notes The authors declare no competing financial interest.

Author Contributions Y.Y. designed the study, analysed the data and wrote the manuscript. H.X.C., X.Y.Y., H.H.X., Y.Y.H., X.R., and J.J.W. performed the experiments. J.L. analysed the data. D.K.Y. and X.D.G. are the guarantors of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. All authors reviewed the manuscript.



References 1. Pandey, M. K.; Karelia, D.; Amin, S. G. Gambogic Acid and Its Role in Chronic Diseases. Adv. Exp. Med. Biol. 2016, 928, 375-395. 2. Kashyap, D.; Mondal, R., Tuli, H. S.; Kumar, G.; Sharma, A. K. Molecular Targets of Gambogic Acid in Cancer: Recent Trends and Advancements. Tumour Biol. 2016, 37, 12915-12925. 3. Wang, X.; Chen, W. Gambogic Acid is a Novel Anti-cancer Agent that Inhibits Cell Proliferation, Angiogenesis and Metastasis. Anticancer Agents Med. Chem.

2012, 12, 994-1000. 4. Gazdar, A. F.; Bunn, P. A.; Minna, J. D. Small-cell Lung Cancer: What We Know, What We Need to Know and the Path Forward. Nat. Rev. Cancer. 2017, 17, 725737. 5. Hirsch, F. R.; Scagliotti, G. V.; Mulshine, J. L.; Kwon, R.; Curran, W. J. Jr.; Wu, Y. L.; Paz-Ares, L. Lung Cancer: Current Therapies and New Targeted Treatments. Lancet. 2017, 389, 299-311. 6. Lu, N.; Yang, Y.; You, Q. D.; Ling, Y.; Gao, Y.; Gu, H. Y.; Zhao, L.; Wang, X. T.; Guo, Q. L. Gambogic Acid Inhibits Angiogenesis through Suppressing Vascular Endothelial Growth Factor-induced Tyrosine Phosphorylation of KDR/Flk-1. Cancer Lett. 2007, 258, 80-89. 7. Lu, N.; Hui, H.; Yang, H.; Zhao, K.; Chen, Y.; You, Q. D.; Guo, Q. L. Gambogic Acid Inhibits Angiogenesis through Inhibiting PHD2-VHL-HIF-1α Pathway. Eur. J. Pharm. Sci. 2013, 49, 220-226. 8. Wen, J.; Pei, H.; Wang, X.; Xie, C.; Li, S.; Huang, L.; Qiu, N.; Wang, W.; Cheng, X.; Chen, L. Gambogic Acid Exhibits Anti-psoriatic Efficacy through Inhibition of Angiogenesis and Inflammation. J. Dermatol. Sci. 2014, 74, 242-250. 9. Yi, T.; Yi, Z.; Cho, S. G.; Luo, J.; Pandey, M. K.; Aggarwal, B. B.; Liu, M. Gambogic Acid Inhibits angiogenesis and Prostate Tumor Growth by Suppressing Vascular Endothelial Growth Factor Receptor 2 Signaling. Cancer Res. 2008, 68, 1843-1850. 10. He, L. Q.; Ling, Y.; Li, F.; Yin, D. K.; Wang, X. S.; Zhang, Y. H. Synthesis and Biological Evaluation of Novel Derivatives of Gambogic Acid as Antihepatocellular Carcinoma Agents. Bioorg. Med. Chem. Lett. 2012, 22, 289-292. 11. Kang, Y.; Lu, L.; Lan, J.; Ding, Y.; Yang, J.; Zhang, Y.; Zhao, Y.; Zhang, T.; Ho, R. J. Y. Redox-responsive Polymeric Micelles Formed by Conjugating Gambogic


Page 18 of 34

Page 19 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Acid with Bioreducible Poly(amido amine)s for the Co-delivery of Docetaxel and MMP-9 shRNA. Acta. Biomater. 2018, 68, 137-153. 12. Yu, F.; Jiang, F.; Tang, X.; Wang, B. N-octyl-N-arginine-chitosan Micelles for Gambogic Acid Intravenous Delivery: Characterization, Cell Uptake, Pharmacokinetics, and Biodistribution. Drug. Dev. Ind. Pharm. 2017, 18, 1-9. 13. Zhang, D.; Zou, Z.; Ren, W.; Qian, H.; Cheng, Q.; Ji, L.; Liu, B.; Liu, Q. Gambogic Acid-loaded PEG-PCL Nanoparticles Act as an Effective Antitumor Agent Against Gastric Cancer. Pharm. Dev. Technol. 2018, 23, 33-40. 14. Yan, X. F.; Yang, Y.; He, L. Q.; Peng, D. Y.; Yin, D. K. Gambogic Acid Grafted Low Molecular Weight Heparin Micelles for Targeted Treatment in a Hepatocellular Carcinoma Model with an Enhanced Anti-angiogenesis Effect. Int. J. Pharm. 2017, 522, 110-118. 15. Zhang, J.; Tang, H.; Liu, Z.; Chen, B. Effects of Major Parameters of Nanoparticles on Their Physical and Chemical Properties and Recent Application of Nanodrug Delivery System in Targeted Chemotherapy. Int. J. Nanomedicine.

2017, 12, 8483-8493. 16. Ventura, C. A.; Tommasini, S.; Crupi, E.; Giannone, I.; Cardile, V.; Musumeci, T.; Puglisi, G. Chitosan Microspheres for Intrapulmonary Administration of Moxifloxacin: Interaction with Biomembrane Models and in Vitro Permeation Studies. Eur. J. Pharm. Biopharm. 2008, 68, 235-244. 17. Yamamoto, H.; Kuno, Y.; Sugimoto, S.; Takeuchi, H.; Kawashima, Y. Surfacemodified PLGA Nanosphere with Chitosan Improved Pulmonary Delivery of Calcitonin by Mucoadhesion and Opening of the Intercellular Tight Junctions. J. Control. Release. 2005, 102, 373-381. 18. Zaru, M.; Manca, M. L.; Fadda, A. M.; Antimisiaris, S. G. Chitosan-coated Liposomes for Delivery to Lungs by Nebulisation. Colloids Surf. B Biointerfaces.

2009, 71, 88-95. 19. Liu, C. Y.; Miao, Y. Q.; Wei, Y. M.; Yang, Z. Z. Establish and Optimization of Fluorescent Labeling of Carboxylfractions in Chinese Medicine. Chem. Res. Appl.

2012, 7, 1030-1035. 20. Yu, F.; He, C.; Waddad, A. Y.; Munyendo, W. L.; Lv, H.; Zhou, J.; Zhang, Q. NOctyl-N-arginine-chitosan (OACS) Micelles for Gambogic Acid Oral Delivery: Preparation, Characterization and its Study on in Situ Intestinal Perfusion. Drug Dev. Ind. Pharm. 2013, 40, 774-782.



21. Gupta, P. K.; Hung, C. T. Quantitative Evaluation of Targeted Drug Delivery Systems. Int. J. Pharm. 1989, 56, 217-226. 22. Liu, J.; Liu, X.; Cui, F.; Chen, G. Q.; Guan, Y. B.; He, J. X. The Efficacy of the Inhalation of an Aerosolized Group A Streptococcal Preparation in the Treatment of Lung Cancer. Chin. J. Cancer Res. 2012, 24, 346-352. 23. Shao, G.; Zhang, R.; Wang, Z. L.; Gao, C. Y.; Huo, X.; Lu, G. W. Hypoxic Preconditioning Improves Spatial Cognitive Ability in Mice. Neurosignals. 2006, 15:314-321. 24. Pan, W.; Hua, X.; Wang, Y.; Guo, R.; Chen, J.; Mo, L. Dose Response of Dexmedetomidine‑induced Resistance to Hypoxia in Mice. Mol. Med. Rep. 2016, 14, 3237-3242. 25. Shen, Z. B.; Yin, Y. Q.; Tang, C. P.; Yan, C. Y.; Chen, C.; Guo, L. B. Pharmacodynamic Screening and Simulation Study of Anti-hypoxia Active Fraction of Xiangdan Injection. J. Ethnopharmacol. 2010, 127, 103-107. 26. Porsolt, R. D.; Le Pichon, M.; Jalfre, M. Depression: A New Animal Model Sensitive to Antidepressant Treatments. Nature. 1977, 266, 730-732. 27. Gerdin, A. K.; Surve, V. V.; Jönsson, M.; Bjursell, M.; Björkman, M.; Edenro, A.; Schuelke, M.; Saad, A.; Bjurström, S.; Lundgren, E. J.; Snaith, M.; FranssonSteen, R.; Törnell, J.; Berg, A. L.; Bohlooly-Y, M. Phenotypic Screening of Hepatocyte Nuclear Factor (HNF) 4-gamma Receptor Knockout Mice. Biochem. Biophys. Res. Commun. 2006, 349, 825-832. 28. Nashed, M. G.; Seidlitz, E. P.; Frey, B. N.; Singh, G. Depressive-like Behaviours and Decreased Dendritic Branching in the Medial Prefrontal Cortex of Mice with Tumors: A Novel Validated Model of Cancer-induced Depression. Behav. Brain Res. 2015, 294, 25-35. 29. Weidner, N. Current Pathologic Methods for Measuring Intratumoral Microvessel Density within Breast Carcinoma and Other Solid Tumors. Breast Cancer Res. Treat. 1995, 36, 169-180. 30. Li, R.; Ren, M. P.; Chen, N.; Luo, M.; Deng, X.; Xia, J. Y.; Yu, G.; Liu, J. B.; He, B.; Zhang, X.; Zhang, Z.; Zhang, X.; Ran, B.; Wu, J. B. Presence of Intratumoral Platelets is Associated with Tumor Vessel Structure and Metastasis. BMC Cancer.

2014, 14: 167.


Page 20 of 34

Page 21 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


31. Yin, D. K.; Yang, Y.; Cai, H. X.; Wang, F.; Peng, D. Y.; He, L. Q. Gambogic Acid-loaded Electrosprayed Particles for Site-specific Treatment of Hepatocellular Carcinoma. Mol. Pharm. 2014, 11, 4107-4117. 32. Yu, D. G.; Williams, G. R.; Wang, X.; Liu, X. K.; Li, H. L.; Blighd, S. W. A. Dual Drug Release Nanocomposites Prepared using a Combination of Electrospraying and Electrospinning. RSC Adv. 2013, 3, 4652-4658. 33. Pittella, F.; Zhang, M.; Lee, Y.; Kim, H. J.; Tockary, T.; Osada, K.; Ishii, T.; Miyata, K.; Nishiyama, N.; Kataoka, K. Enhanced Endosomal Escape of siRNAincorporating Hybrid Nanoparticles from Calcium Phosphate and PEG-block Charge-conversional Polymer for Efficient Gene Knockdown with Negligible Cytotoxicity. Biomaterials. 2011, 32, 3106-3114. 34. Tang, J.; Li, L.; Howard, C. B.; Mahler, S. M.; Huang, L.; Xu, Z. P. Preparation of Optimized Lipid-coated Calcium Phosphate Nanoparticles for Enhanced in Vitro Gene Delivery to Breast Cancer Cells. J. Mater. Chem. B Mater. Biol Med. 2015, 3, 6805-6812. 35. Zahr, A. S.; Davis, C.A.; Pishko, M. V. Macrophage Uptake of Core-shell Nanoparticles Surface Modified with Poly(ethylene glycol). Langmuir. 2006, 22, 8178-8185. 36. Shankar, A.; Dracham, C.; Ghoshal, S.; Grover, S. Prevalence of Depression and Anxiety Disorder in Cancer Patients: An Institutional Experience. Indian J. Cancer. 2016, 53, 432-434. 37. Amirifard, N.; Payandeh, M.; Aeinfar, M.; Sadeghi, M.; Sadeghi, E.; Ghafarpor, S. A Survey on the Relationship between Emotional Intelligence and Level of Depression and Anxiety among Women with Breast Cancer. Int. J. Hematol. Oncol. Stem Cell Res. 2017, 11, 54-57. 38. Dimsdale, J.; Creed, F.; Escobar, J.; Sharpe, M.; Wulsin, L.; Barsky, A.; Lee, S.; Irwin, M. R.; Levenson, J. Somatic Symptom Disorder: an Important Change in DSM. J. Psychosom. Res. 2013, 75, 223-228. 39. Huang, Y.; Song, N.; Ding, Y.; Yuan, S.; Li, X.; Cai, H.; Shi, H.; Luo, Y. Pulmonary Vascular Destabilization in the Premetastatic Phase Facilitateslung Metastasis. Cancer Res. 2009, 69, 7529-7537. 40. Zhou, W.; Fong, M. Y.; Min, Y.; Somlo, G.; Liu, L.; Palomares, M. R.; Yu, Y.; Chow, A.; O'Connor, S. T.; Chin, A. R.; Yen, Y.; Wang, Y.; Marcusson, E. G.; Chu, P.; Wu, J.; Wu, X.; Li, A. X.; Li, Z.; Gao, H.; Ren, X.; Boldin, M. P.; Lin, P.



C.; Wang, S. E. Cancer-secreted miR-105 Destroysvascular Endothelial Barriers to Promote Metastasis. Cancer Cell. 2014, 25, 501-515. 41. Gupta, G. P.; Nguyen, D. X.; Chiang, A. C.; Bos, P. D.; Kim, J. Y.; Nadal, C.; Gomis, R. R.; Manova-Todorova, K.; Massagué, J. Mediators of Vascular Remodeling co-opted for Sequential Steps in Lung Metastasis. Nature. 2007, 446, 765-770. 42. Matt, L.; Sehgal, R. Metastatic Non-small-cell Lung Cancer to the Liver and Pancreas. Gastrointest Cancer Res. 2014, 7, 61-62. 43. Castañón, E.; Rolfo, C.; Viñal, D.; López, I.; Fusco, J. P.; Santisteban, M.; Martin,

P.; Zubiri, L.; Echeveste, J. I.; Gil-Bazo, I. Impact of Epidermal Growth Factor Receptor (EGFR) Activating Mutations and Their Targeted Treatment in the Prognosis of Stage IV Non-small Cell Lung Cancer (NSCLC) Patients Harboring Liver Metastasis. J. Transl. Med. 2015, 13, 257.


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Figure 1. (a) Chemical structure of GA, and (b) size distribution, (c) representative SEM image and (d) in vitro drug release profile of GA-loaded particle CS/PLGA-GA.



Figure 2. (a) Drug concentration (a1, group of GA-S; a2, group of GA-P) and (b) drug targeting index (DTI) of GA in tissues of the heart, liver, spleen, lung, and kidney, after tail-vein injection of GA solution and GA-loaded particle of CS/PLGAGA at a dosage of 2.5 mg/kg. (c) Fluorescent microscopy images of tissues in mice administered with ATPS-GA solution at 5 min post-dose and ATPS-GA loaded particle at 10 min post-dose.


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Figure 3. (a) Standard hypoxic tolerance time (Ts, a1) and representative experiment picture (a2) of the hypoxic tolerance time test on LLC mice after administration of normal saline (NS group), GA solution (GA-S group), or GA-loaded particle of CS/PLGA-GA (GA-P group), compared with normal mice (Normal group) and LLC model mice without any administration (Model group). (b) Bodyweight curves of LLC mice in groups of model, NS, GA-S, and GA-P, with normal mice as control. (c) Swimming time (c1) and representative experiment picture (c2) of the porsolt forced swim test on LLC mice in groups of model, NS, GA-S, and GA-P, with normal mice as control. (d) The survival time of LLC mice in groups of model, NS, GA-S, and GA-P. *, p < 0.05 vs normal group; #, p < 0.05 vs model group and NS group; &, p < 0.05 vs GA-S group.



Figure 4. Pulmonary histomorphology of LLC mice, at the seventh day after tail vein injection of normal saline (NS group, b), GA-solution (GA-S group, c), and GAloaded particle of CS/PLGA-GA (GA-P group, d), compared with model mice without any administration (Model group, a). Samples are stained with HE. (AS, alveolar septa; A, alveoli; TB, terminal bronchiole; black arrow, simple cuboidal or columnar epithelium; yellow arrow, tumour cells.)


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Figure 5. (a) Representative IHC staining pictures of CD31 (a1), relative CD31 levels, and microvessel density (a3) in lung tissues of LLC mice, at the seventh day after tail vein injection of normal saline (NS group), GA-solution (GA-S group), and GAloaded particle of CS/PLGA-GA (GA-P group), compared with model mice without any administration(Model group). (b) Representative electron microscopy image of the capillary in lung tissue of LLC model mice. The gaps in the wall of the tinny



capillary are indicated with a red dashed line. (c) Absorbance ratios of perfusates to serum in LLC mice for pulmonary vascular permeability test, at the seventh day after tail vein injection of normal saline (NS group), GA-solution (GA-S group), and GAloaded particle of CS/PLGA-GA (GA-P group), compared with normal mice (Normal group) and LLC model mice without any administration (Model group). *, p < 0.05 vs normal group; #, p < 0.05 vs model group and NS group; &, p < 0.05 vs GA-S group.


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Figure 1. (a) Chemical structure of GA, and (b) size distribution, (c) representative SEM image and (d) in vitro drug release profile of GA-loaded particle CS/PLGA-GA. 177x142mm (300 x 300 DPI)



Figure 2. (a) Drug concentration (a1, group of GA-S; a2, group of GA-P) and (b) drug targeting index (DTI) of GA in tissues of the heart, liver, spleen, lung, and kidney, after tail-vein injection of GA solution and GAloaded particle of CS/PLGA-GA at a dosage of 2.5 mg/kg. (c) Fluorescent microscopy images of tissues in mice administered with ATPS-GA solution at 5 min post-dose and ATPS-GA loaded particle at 10 min postdose. 177x185mm (300 x 300 DPI)


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Figure 3. (a) Standard hypoxic tolerance time (Ts, a1) and representative experiment picture (a2) of the hypoxic tolerance time test on LLC mice after administration of normal saline (NS group), GA solution (GA-S group), or GA-loaded particle of CS/PLGA-GA (GA-P group), compared with normal mice (Normal group) and LLC model mice without any administration (Model group). (b) Bodyweight curves of LLC mice in groups of model, NS, GA-S, and GA-P, with normal mice as control. (c) Swimming time (c1) and representative experiment picture (c2) of the porsolt forced swim test on LLC mice in groups of model, NS, GA-S, and GAP, with normal mice as control. (d) The survival time of LLC mice in groups of model, NS, GA-S, and GA-P. *, p < 0.05 vs normal group; #, p < 0.05 vs model group and NS group; &, p < 0.05 vs GA-S group. 181x140mm (300 x 300 DPI)



Figure 4. Pulmonary histomorphology of LLC mice, at the seventh day after tail vein injection of normal saline (NS group, b), GA-solution (GA-S group, c), and GA-loaded particle of CS/PLGA-GA (GA-P group, d), compared with model mice without any administration (Model group, a). Samples are stained with HE. (AS, alveolar septa; A, alveoli; TB, terminal bronchiole; black arrow, simple cuboidal or columnar epithelium; yellow arrow, tumour cells.) 174x45mm (300 x 300 DPI)


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Figure 5. (a) Representative IHC staining pictures of CD31 (a1), relative CD31 levels, and microvessel density (a3) in lung tissues of LLC mice, at the seventh day after tail vein injection of normal saline (NS group), GA-solution (GA-S group), and GA-loaded particle of CS/PLGA-GA (GA-P group), compared with model mice without any administration(Model group). (b) Representative electron microscopy image of the capillary in lung tissue of LLC model mice. The gaps in the wall of the tinny capillary are indicated with a red dashed line. (c) Absorbance ratios of perfusates to serum in LLC mice for pulmonary vascular permeability test, at the seventh day after tail vein injection of normal saline (NS group), GA-solution (GA-S group), and GA-loaded particle of CS/PLGA-GA (GA-P group), compared with normal mice (Normal group) and LLC model mice without any administration (Model group). *, p < 0.05 vs normal group; #, p < 0.05 vs model group and NS group; &, p < 0.05 vs GA-S group. 181x243mm (300 x 300 DPI)




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Table of Contents/Abstract Graphic 88x35mm (300 x 300 DPI)


Efficient Targeting Drug Delivery System for Lewis Lung Carcinoma

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