Endotracheal Aerosolization Device for Laboratory ... - ACS Publications

Subscriber access provided by Kaohsiung Medical University

Article

An Endotracheal Aerosolization Device for Laboratory Investigation of Pulmonary Delivery of Nanoparticle Suspensions: in vitro and in vivo Validation Zhengwei Huang, Ying Huang, Cheng Ma, Xiangyu Ma, Xuejuan Zhang, Ling Lin, Ziyu Zhao, Xin Pan, and Chuanbin Wu Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.8b00668 • Publication Date (Web): 25 Sep 2018 Downloaded from http://pubs.acs.org on September 29, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 42 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

Molecular Pharmaceutics

An Endotracheal Aerosolization Device for Laboratory Investigation of Pulmonary Delivery of Nanoparticle Suspensions: in vitro and in vivo Validation Zhengwei Huanga, Ying Huanga,*, Cheng Maa, Xiangyu Mab, Xuejuan Zhanga,c, Ling Lina, Ziyu Zhaoa, Xin Pana,**, Chuanbin Wua a

School of Pharmaceutical Sciences, Sun Yat-Sen University, Guangzhou 510006, Guangdong,

P. R. China b

College of Pharmacy, The University of Texas at Austin, Austin, TX 78712, USA

c

Institute for Biomedical and Pharmaceutical Sciences, Guangdong University of Technology,

Guangzhou, 510006, P.R. China

* and ** are corresponding authors. Tel: +86 02039943115 (Ying Huang, Ph.D.), +86 02039943427 (Xin Pan, Ph.D.) Fax: +86 02039943115 (Ying Huang, Ph.D.), +86 02039943117 (Xin Pan, Ph.D.)

E-mail addresses of the authors are listed below. Zhengwei Huang (Z. Huang, Z. H.): [email protected] Ying Huang (Y. Huang, Y. H.): [email protected] Cheng Ma (C. Ma, C. M.): [email protected] Xiangyu Ma (X. Ma, X. M.): [email protected] Xuejuan Zhang (X. Zhang, X. Z.): [email protected] Ling Lin (L. Lin, L. L.): [email protected] Ziyu Zhao (Z. Zhao, Z. Z.): [email protected] Xin Pan (X. Pan, X. P.): [email protected] Chuanbin Wu (C. Wu, C. W.): [email protected]

1

ACS Paragon Plus Environment

Molecular Pharmaceutics 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

1

An Endotracheal Aerosolization Device for Laboratory Investigation of

2

Pulmonary Delivery of Nanoparticle Suspensions: in vitro and in vivo Validation

3

Abstract:

4

The objective of this study was to perform the in vitro and in vivo validation of an endotracheal

5

aerosolization (ETA) device (HRH MAG-4, HM). Solid lipid nanoparticle suspension (SLNS)

6

formulations with particle sizes of approximately 120, 240, 360 and 480 nm were selected as

7

model nanoparticle suspensions for the validation. The emission rate (ER) of the in vitro

8

aerosolization and the influence of aerosolization on the physicochemical properties were

9

investigated. A high ER of up to 90% was obtained, and no significant alterations in

10

physicochemical properties were observed after the aerosolization. The pulmonary deposition of

11

model drug budesonide in Sprague-Dawley rats was determined to be approximately 80%, which

12

was satisfactory for pulmonary delivery. Additionally, a fluorescent probe with aggregation-caused

13

quenching property was encapsulated in SLNS formulations for in vivo bioimaging, after

14

excluding the effect of aerosolization on its fluorescence spectrum. It was verified that SLNS

15

formulations were deposited in the lung region. The results demonstrated the feasibility and

16

reliability of the HM device for ETA in laboratory investigation.

17

Keywords:

18

Pulmonary delivery; nanoparticle; aerosolization device; aggregation-caused quenching;

19

bioimaging

20

1


Page 2 of 42

Page 3 of 42 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


21

1 Introduction

22

Due to their behavior that results in escape from the endosomes in the lung region, a phenomenon

23

termed “nanoescapology” 1, nanoparticles are considered to be a promising vehicle for pulmonary

24

drug delivery. The application of two kinds of nanoparticles for pulmonary drug delivery has been

25

reported: solid nanoparticles (solid state as a dry powder) and nanoparticle suspensions (mainly

26

referred to aqueous suspensions). Generally, solid nanoparticles are employed

27

inhalers 2, whereas nanoparticle suspensions can be administered using nebulizers 3 or sprayers 4.

28

The authors believe that nanoparticle suspensions are the preferred delivery vehicles. This is

29

because most nanoparticles are fabricated in liquid media

30

in situ delivery platform can prevent the particle-aggregation issues associated with the

31

solidification process in solid nanoparticle preparation 7, 8. Until now, the laboratory investigation

32

of nanoparticle suspensions for pulmonary delivery has gained extensive interest among

33

researchers,

34

pharmacodynamic profiles have been demonstrated 9, 10.

35

However, the emphasis of previous laboratory investigation has been the preclinical outcome, and

36

fewer studies are focused on the pulmonary delivery approach of nanoparticle suspensions. This

37

omission may lead to possible artifacts, since different pulmonary delivery approaches will result

38

in different in vivo pharmacokinetic and pharmacodynamic profiles 11. A proper approach should

39

be unambiguously determined for the laboratory investigation to ensure the lack of such artifacts.

40

It is noteworthy that the choices of methods for pulmonary delivery of nanoparticle suspensions in

41

animals is limited, which remains a challenge that must be met 12. Active inhalation is impractical

42

in animal models, and semipassive/passive inhalation methods such as nebulization

and

many

exciting

results

5, 6

including

dry powder

, and nanoparticle suspensions as an

improved

2


pharmacokinetic

and

13

,


14

15

Page 4 of 42

16

43

oropharyngeal aspiration

44

endotracheal aerosolization (ETA) are available methods. Nevertheless, some critical

45

shortcomings are associated with these methods, except for ETA. Dose inaccuracy is often

46

observed in nebulization, oropharyngeal aspiration and metered-dose inhalation as the result of

47

dose wastage. With the help of a nebulizer, nanoparticle suspensions can be nebulized into the air

48

and the drug-containing mist can be inhaled by the animals

49

nebulized nanoparticle suspensions will remain in the atmosphere or may attach to the skin of

50

animals and are therefore not inhaled 18. Oropharyngeal aspiration refers to a procedure in which

51

the nanoparticle suspensions are placed in the oropharyngeal region and inhaled by reflex 19. The

52

administered nanoparticle suspensions may be swallowed and transported into the gastrointestinal

53

tract after oropharyngeal aspiration, which reduces the amount of nanoparticle suspensions that

54

enter the pulmonary region

55

method, where nanoparticle suspensions are delivered with the propellant on actuation

56

However, actuation-inhalation-coordination

57

exhalation or escape of the delivered nanoparticle suspensions and results in a low pulmonary

58

deposition. For intratracheal instillation, the trachea is exposed and nanoparticle suspensions are

59

directly instilled into the respiratory tract

60

achieved, the surgery that is performed to expose the tracheal region causes relatively severe

61

wounds 11. These limitations may influence the laboratory investigation of pulmonary delivery of

62

nanoparticle suspensions in animals.

63

In ETA, nanoparticle suspensions are directly aerosolized within the trachea and readily deposited

64

into the pulmonary region 24. A more satisfactory dose accuracy can be assured in ETA compared

, metered-dose inhalation

20

, intratracheal instillation

17

and

. A considerable amount of the

. Metered-dose inhalation is an actuation-dependent administration

22

23

21

.

cannot be assured in animals, which leads to

. Although a satisfactory dose accuracy can be

3


Page 5 of 42 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


65

to nebulization, oropharyngeal aspiration and metered-dose inhalation 12 because theoretically the

66

entire dose of nanoparticle suspensions will be located in the trachea. Additionally, ETA is a

67

noninvasive delivery method (if performed with the assistance of a laryngoscope), which is a more

68

physiological intervention than intratracheal instillation

69

promising method for the pulmonary delivery of nanoparticle suspensions in animals.

70

It is worth mentioning that the success of ETA administration is dependent on the ETA device. A

71

qualified ETA device should deliver the loaded nanoparticle suspensions with a high efficiency

72

and should not alter the physicochemical properties of nanoparticle suspensions. There was only

73

one robust and widely accepted ETA device on the market, Microsprayer™ 25, and regrettably, its

74

production ceased in 2015. HRH MAG-4 (HM, Huironghe Technology Co., Ltd. Beijing, China)

75

is a recently developed ETA device for rats. It is designed to assist the laboratory pharmacokinetic

76

and pharmacodynamic investigation of nanoparticle suspensions for rodent models. According to

77

the clarification of the manufacturer, two characteristics differ between HM and Microsprayer™:

78

(I) the inner diameter of the delivery channel of HM is larger than that of Microsprayer™. This

79

means that samples with a larger particle size can be loaded and delivered, and a larger inner

80

diameter will result in a lower aerosolization pressure (Bernoulli principle

81

body of HM is smaller than that of Microsprayer™, which facilitates its manual use. Nevertheless,

82

no publications up to the present have verified the feasibility and reliability of HM. The ability of

83

HM to deliver a nanoparticle suspension with a satisfactory efficiency is uncertain, due to the

84

lower aerosolization pressure of HM. Furthermore, whether ETA by HM will influence the

85

physicochemical properties of nanoparticle suspensions remains unknown. These uncertainties

86

limit the further application of HM as an emerging alternative for Microsprayer™ for academic

11

. Hence, ETA is an appropriate and

4


26

) and (II) the main


87

and industrial use.

88

This work was aimed to perform the in vitro and in vivo validation of HM. Solid lipid nanoparticle

89

suspensions (SLNS) were prospective nanoparticulate vehicles for pulmonary delivery due to their

90

high safety and deep lung deposition 27. Therefore, SLNS was selected as the model nanoparticle

91

suspension. The physicochemical and morphological properties of the SLNS formulations were

92

investigated, and in vitro aerosolization and ETA were conducted by HM. The in vitro emission

93

ratio, influence of aerosolization on SLNS physicochemical properties and sample residue were

94

investigated in detail. Further, in vivo pulmonary deposition and bioimaging were performed.

95

Noticeably, a versatile aggregation-caused quenching (ACQ) fluorescent probe with an

96

aza-BODIPY fluorophore

97

that the validation of HM could be accomplished by these in vitro and in vivo tests.

28-30

was employed in the bioimaging examination. It was anticipated

98

99

2 Materials and methods

100

2.1 Materials

101

Palmityl palmitate (PP) and dichloromethane (DCM) were purchased from Aladdin Industrial Inc.

102

(Shanghai, China). Polysorbate 80 (Tween 80), ethanol absolute and ethyl acetate were obtained

103

from Damao Chemical Reagent Factory (Tianjin, China). Budesonide (BUD) was supplied by

104

Hubei Jusheng Technology Co., Ltd. (Hubei, China). Triamcinolone acetonide (TAA), the internal

105

standard for the in vivo studies, was obtained from National Institutes for Food and Drug Control

106

(Beijing, China). Acetonitrile (HPLC grade) was purchased from Honeywell International Inc.

107

(Morristown, NJ). Ultrapure water was obtained using the PureLAB option water purification 5


Page 6 of 42

Page 7 of 42 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


108

system (ELGA Lab Water Inc., High Wycombe, United Kingdom).

109

The ACQ probe, coded P4, was designed and synthesized by Wei Wu et al. in Fudan University

110

(Shanghai, China) according to the published method 31. Detailed information regarding P4 can be

111

found in the literature 32, 33.

112

2.2 Animals

113

The animals involved in this study were Sprague-Dawley (SD) rats. Male SD rats weighing

114

approximately 180 g were supplied by Guangdong Medical Laboratory Animal Center

115

(Guangdong, China). The SD rats were housed 5 per cage under a condition of 23 ± 2°C, 55 ± 5%

116

relative humidity and noise ≤ 60 dB. Food and water were provided ad libitum until one day

117

before dosing. All protocols were approved by the Animal Ethical and Welfare Committee at Sun

118

Yat-sen University (approval No. IACUC-DD-17-1108).

119

2.3 Pretreatment of PP for the fabrication of SLNS

120

PP, the lipid employed for SLNS fabrication, was pretreated. The PP was successively washed

121

with water and ethanol absolute, and then vacuum-dried under 25°C (Bluepard® BPZ-6210-2,

122

Shanghai Yi Heng Scientific Instruments Co., Ltd.). The washed PP was melted using a 65°C

123

water bath and immediately filtered through a preheated 0.22 µm nylon-66 membrane (Membrana

124

GmbH, Wuppertal, Germany). The filtrate was vacuum-dried at 25°C, and the obtained PP was

125

collected and stored in a sealed container in a dark environment at 20°C.

126

2.4 Production of SLNS

127

PP was melted using a water bath (lipid phase), and 50 mL of an aqueous solution of Tween 80 6



128

was heated to the same temperature (aqueous phase). For the BUD- or P4-containing formulations,

129

1.5 mL of BUD or P4 stock solution was added to the melted PP. These stock solutions were

130

prepared by dissolving BUD or P4 in DCM at concentrations of 40 µg/mL or 100 µg/mL,

131

respectively. The DCM in the lipid phase was allowed to evaporate while the solution was stirred.

132

The lipid phase was then poured into the aqueous phase, and the system was subjected to

133

high-shear emulsification (FA25, FLUKO Equipment Shanghai Co., Ltd.). The obtained crude

134

emulsion was subjected to hot high-pressure homogenization (EmusiFlex-C3, Avestin Inc., Ottawa,

135

Canada). Three batches of each SLNS formulation were produced. A scheme of the preparation

136

procedure is provided in Fig. 1A. The related formulation details and processing parameters are

137

summarized in Tab. 1. For simplicity, the blank, BUD-loaded, and P4-loaded SLNS formulations

138

were coded as b-SLNS, BUD-SLNS and P4-SLNS, respectively; the SLNS with different particle

139

sizes were denoted as SLNS1~SLNS4.

140

(Please insert Fig. 1 here.)

141

(Please insert Tab. 1 here.)

142

2.5 Physicochemical characterization of SLNS

143

2.5.1 Particle size analysis and zeta-potential (ZP) examination

144

The particle size and ZP of the SLNS were determined using a Zetasizer Nano ZS90 (Malvern

145

Instruments Ltd., Worcestershire, UK). The SLNS was diluted by 120-fold with ultrapure water

146

prior to the tests to ensure a proper count rate (circa 300 kcps). The particle size and ZP were

147

analyzed at 37°C. Ten scans and twenty scans were performed for each measurement of the

148

particle size analysis and ZP determination, respectively, and three parallel measurements were

7


Page 8 of 42

Page 9 of 42 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


149

conducted.

150

2.5.2 Density (ρ) determination

151

Ultrasonication (15 s) was performed on the SLNS formulations prior to ρ determination. One

152

hundred microliters of the SLNS formulations was precisely weighed, and ρ was calculated by the

153

weight divided by the volume. The measurement was performed in triplicate.

154

2.5.3 Drug loading (DL) and encapsulation efficiency (EE) determinations

155

The prepared SLNS consisted of the suspended nanoparticles and the bulk aqueous phase

156

certain fraction of the drugs would be distributed in the bulk aqueous phase because of loading

157

failure and/or leakage of nanoparticles during the fabrication and storage process 35. Thus, the DL

158

and EE of both the suspended nanoparticles (termed as DLSLN and EESLN) and the entire SLNS

159

formulations consisting of the suspended nanoparticles and the aqueous phase (DLSLNS and EESLNS)

160

were determined. Ultrafiltration tubes (Merck KGaA, Darmstadt, Germany) were employed in the

161

assay. The details of the measurements of the DLSLN, EESLN, DLSLNS and EESLNS can be found in

162

Supporting information Section 1.

163

2.5.4 Dynamic viscosity evaluation

164

The dynamic viscosity of b-SLNS, BUD-SLNS and P4-SLNS was investigated using a Kinexus

165

Lab+ Rheometer (Malvern Instruments Ltd., Worcestershire, UK). For each sample, approximately

166

4 mL was applied to the sample stub. The viscosity was recorded within the shear rate of 10~100

167

s-1 at 25°C according to a standard operation procedure provided by the rSpace operation software

168

(Malvern Instruments Ltd., Worcestershire, UK). The cone used in the tests was CP 4/60 (60 mm

8


34

.A


169

diameter, 4° cone angle and 0.03 mm gap truncation).

170

2.6 Transmission electron microscopy (TEM)

171

The freshly produced SLNS formulations, including b-SLNS1~4, BUD-SLNS1~4 and

172

P4-SLNS1~4, were subjected to examination by TEM. The samples were carefully placed on

173

copper grids and then negatively stained with a 1% (w/v) phosphotungstic acid solution. The

174

excess solution was absorbed prior to being examined by a 100 kV JEM-100 CX II transmission

175

electron microscope (JEOL Ltd., Tokyo, Japan).

176

2.7 In vitro and in vivo validation of the HM device

177

The HM device was utilized for the in vitro aerosolization and ETA of SLNS. Detailed

178

information about the composition and application of HM was as follow.

179

A typical image of HRH-MAG4 is shown in Fig. 1B. It included four parts, i.e., a delivery tube (I),

180

a micronebulizer (II), a sample cell (III) and a piston (IV), with an accessory volume-fixer (V).

181

The delivery tube was placed in the liquid sample, and then the piston was used to generate a

182

difference in pressure for sample loading. Ultrasonication (15 s) was performed on the samples

183

prior to sample loading. The piston was held for at least 15 s for a sufficient loading. Three

184

volume-fixers were installed on the rod of the piston. One, two or three volume-fixers could be

185

removed in order to aerosolize 50, 100 or 150 µL of sample (50 µL per volume-fixer).

186

2.7.1 In vitro emission ratio (ER) determination

187

The weight of 50 µL of each sample (m), viz. b-SLNS, BUD-SLNS and P4-SLNS, was calculated

188

based on the determined ρ values. Subsequently, 150 µL samples were successively loaded into

9


Page 10 of 42

Page 11 of 42 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


189

the HM. The initial weight of loaded device was denoted as m150. Eppendorf tubes with the

190

opening covered with paraffin film were used as the receptor for the aerosolized samples. The

191

paraffin film was carefully penetrated by the delivery tube of HM, and continuous aerosolization

192

was performed in one step. After the in vitro aerosolization of the 50, 100 and 150 µL samples, the

193

device was weighed, and the weights were recorded as m100, m50 and m0. The ER for each

194

aerosolization volume (ER50, ER100 and ER150) was obtained by Eq. 1:

195

ER =

× 100% Eq. 1

196

where ERx represented ER50, ER100 or ER150. For ER50, ER100 or ER150, my = m100, m50 or m0,

197

respectively.

198

2.7.2 Influence of in vitro aerosolization on the physicochemical properties of the SLNS

199

formulations

200

The formulations of the SLNS that had been aerosolized in vitro were carefully collected. Their

201

physicochemical properties including particle size distribution, ZP, DL and EE were evaluated as

202

described previously. Negligible influence of aerosolization by HM could be confirmed if no

203

distinct change in physicochemical properties was observed.

204

2.7.3 Influence of in vitro aerosolization on the fluorescence spectrum of P4

205

The P4-SLNS formulations before and after in vitro aerosolization were dissolved in acetonitrile

206

in the 70°C water bath. Sample dissolution was accomplished within a sealed container to avoid

207

evaporation of the acetonitrile. The resulting solution was filtered through a 0.22 µm nylon-66

208

membrane, and the fluorescence spectrum was scanned (Fluoromax-4, Horiba Instruments Inc.,

209

Albany, NY) under ambient conditions. The scanning was carried out using a slit-width of 2 nm.

10



210

The excitation wavelength was set at 651 nm, and the emission wavelength range was 350~850

211

nm with 1 nm step-size. An acetonitrile solution of P4 was selected as a reference for this test.

212

2.7.4 Sample residues assessment

213

The residual amount of BUD-SLNS and P4-SLNS in the sample cell of HM was quantified. After

214

in vitro aerosolization, the device was rinsed thoroughly with 10 mL of hot ethanol absolute (c.a.

215

70°C). The resulting solution was vacuum-dried, and the residue was reconstituted in 200 µL hot

216

acetonitrile (c.a. 70°C). Sample dissolution was completed within a sealed container to avoid

217

evaporation of the acetonitrile. The supernatant was filtered through a 0.22 µm nylon-66

218

membrane and then assayed. Thereafter, the processed device was extracted by ethanol absolute

219

for a second time and chromatographically quantified, to determine whether any residue remained

220

after one time of washing-off.

221

2.7.5 In vivo pulmonary deposition determination

222

BUD-SLNS1~BUD-SLNS4 were administered to SD rats (n = 5). Briefly, SD rats were

223

anesthetized with 5 mL/kg 20% urethane (w/v) i.p. and immobilized before ETA. The trachea was

224

visualized with the help of a mini laryngoscope for rats (Huironghe Technology Co., Ltd. Beijing,

225

China). The delivery tube of HM (BUD-SLNS loaded) was carefully introduced into the trachea

226

anterior to the first bifurcation. The BUD-SLNS formulations (100 µL) were aerosolized into the

227

respiratory tract of the SD rats. The animals were immediately sacrificed by cervical dislocation,

228

and the lungs were surgically excised. The visible bronchi were carefully removed.

229

The BUD in harvested lungs was assayed as described in Section 2.7. Based on the determined

230

pulmonary BUD concentration, dose recovery was calculated, including total recovery and 11


Page 12 of 42

Page 13 of 42 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


231

emission recovery: !" #$% &'()* +) *, -().

232

Total recovery % =

233

Emission recovery % =

/&"" #$% &'()*

× 100% Eq. 2

!" #$% &'()* +) *, -(). 4'+**" #$% &'()*

× 100% Eq. 3

234

2.7.6 Fluorescence bioimaging study

235

Fluorescence bioimaging was conducted using a NightOWL II LB983 instrument (Berthold

236

Technologies GmbH & Co. KG, Bad Wildbad, Germany). Photographic images of the SD rats

237

before and after ETA were acquired. For the in vivo bioimaging, the SD rats were administered the

238

P4-SLNS1~P4-SLNS4 by the same method in Section 2.6.4. The abdominal hair of SD rats was

239

removed by hair removal cream (Veet, Reckitt Benckiser. Group plc., Hubei, China) to reduce the

240

autofluorescence. The lungs of the administrated SD rats were excised and ex vivo bioimaging was

241

performed. The parameters for the bioimaging were set as follows: exposure time 0.25 s for in

242

vivo bioimaging and 0.10 s for ex vivo bioimaging, high gain (feedback) for in vivo imaging and

243

low gain (feedback) for ex vivo imaging, excitation wavelength 630 nm, emission wavelength 680

244

nm, sample size 160 mm and sample height 22 mm. The quantification of fluorescence signal was

245

conducted using the instrument-associated Indigo™ software.

246

2.8 Chromatography

247

2.8.1 Instrumentation and HPLC conditions

248

Quantification of BUD and P4 was performed using a Dionex Ultimate 3000 instrument (Dionex

249

Solution GmbH, Germering, Germany) that was equipped with a variable wavelength detector

250

(VWD). A Luna C18 column (250 mm × 4.6 mm, 5 µm, Phenomenex Inc., Torrance, CA) was

12



251

utilized for separation. The mobile phase was water-acetonitrile (40:60, v/v) for BUD and

252

water-acetonitrile (18:82, v/v) for P4. The detection wavelengths for BUD and P4 were 240 nm

253

and 651 nm, respectively. The flow rate was set to 1.0 mL/min, and the injection volume was 20

254

µL. The validation of this method is shown in Tab. S1 and S2.

255

2.8.2 Pretreatment of lung samples containing BUD

256

The harvested lungs were washed with cold saline to remove the contaminating blood. Then, the

257

lungs were cut into pieces and saline (5 mL per 1 g of lung) was added. The suspended tissue was

258

then homogenized (IKA®-Werke GmbH, Deutschland, Germany). The resulting homogenate was

259

pretreated as follows:

260

Three hundred microliter of acetic acid (0.1 mg/mL) was added to 1 mL of the homogenate for

261

deproteinization. The solution was then spiked with the internal standard, 200 µL of a 5 µg/mL

262

TAA solution in methanol. The BUD was extracted twice with 2 mL of ethyl acetate. The

263

supernatant was collected after 5000 r/min centrifugation for 15 min at 4°C (GL-20C, Anting

264

Scientific Instrument Factory, Shanghai, China), and then vacuum-dried. The residue was

265

reconstituted with 250 µL of the mobile phase. Finally, the solution was filtered through a 0.22 µm

266

nylon-66 membrane and assayed. Vortex-mixing was conducted when necessary.

267

2.9 Data expression and statistics

268

The data are reported as the means ± SD where applicable. If necessary, grouped data were

269

analyzed by one-way ANOVA with Bonferroni multiple comparisons using SPSS 19.0 software

270

(IBM Corporation, Armonk, NY). In all cases, p < 0.05 was recognized as statistically significant.

13


Page 14 of 42

Page 15 of 42 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


271

3 Results and discussion

272

ETA was an appropriate approach for the pulmonary delivery of nanoparticles in animals.

273

Validation of HM, a newly developed ETA device, was required before its application for ETA. To

274

this end, a reproducible model nanoparticle suspension should first be obtained. Otherwise, the

275

data would not be reliable with a highly variable formulation

276

generate a qualified model nanoparticle suspension, SLNS. After their preparation and

277

physicochemical characterization, SLNS formulations were subjected to the in vitro and in vivo

278

assessment for the validation of the ETA device.

279

3.1 Preparation and characterization of SLNS formulations

280

3.1.1 Preparation

281

Various lipid materials had been used in the fabrication of solid lipid nanoparticles, for example,

282

triglycerides, partial glycerides, fatty acids, long-chain alcohols, steroids and waxes

283

which was categorized as a wax, was one of the most widely investigated and applied lipid

284

materials

285

physical stability

286

present study.

287

PP was pretreated prior to the SLNS production. The purpose of the pretreatment was to remove

288

the impurities that were not soluble in the melted PP and might be responsible for inducing a

289

gelation phenomenon

290

S1). Additionally, the yield of pretreatment was circa 95%.

291

SLNS formulations were produced via the hot high-pressure homogenization method. It has been

39-41

36

. Therefore, the first task was to

on the basis of its low in vivo toxicity, suitable degradation rate 43

42

37, 38

. PP,

and satisfactory

. Therefore, PP was chosen as the lipid material for SLNS production in the

44

. The pretreatment effectively eliminated the gelation phenomenon (Fig.

14



Page 16 of 42

292

reported that nanoparticles with a reproducible particle size could be manufactured using this

293

method

294

method was satisfactory on the basis of the relatively low variability of the parameters.

45

. The characterization results are shown in Fig. 2. Overall, the reproducibility of this

295


296

3.1.2 Characterization

297

The various particle sizes of the SLNS formulations (120, 240, 360 and 480 nm) were generated

298

by adjusting the formulation composition. Specifically, a higher PP-Tween 80 ratio resulted in a

299

larger particle size. This was consistent with the fact that a higher lipid content

300

surfactant content 48 in the solid lipid nanoparticle formulation resulted in larger particle size.

301

The particle size distribution of all formulations was narrow, and all of the PdI (polydispersity

302

index) values were less than 0.25. The use of ultra-pure water for the sample dilution might

303

account for the relatively high deviation of PdI: the system was in the lack of ions to shield the

304

particle-particle interaction might have resulted in a variable hydrodynamic radius

305

formulations demonstrated a negative ZP, which was often reported for lipid-based nanoparticles

306

50-52

307

as-prepared nanoparticles, and since Tween 80 was an electrically neutral compound, the PP was

308

the major contributor to the negative ZP 53. From this viewpoint, the relatively lower absolute ZP

309

value (< 20 mV) of SLNS1 compared to SLNS2~SLNS4 could be explained by the higher content

310

of Tween 80. Moreover, the encapsulation of BUD/P4 exerted negligible impacts on the particle

311

size distribution and ZP of the SLNS formulations (p > 0.05, Fig. 2).

312

The DLSLN and EESLN values ranged between 8.70 and 50.90 ppm (i.e., 0.00087~0.00509%, w/w)

46, 47

49

and lower

. All SLNS

. It could be inferred that PP and Tween 80 were mutually distributed on the surface of the

15


Page 17 of 42 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


313

and 52.74 and 91.41%, respectively, and the values for BUD-SLNS and P4-SLNS were similar (p >

314

0.05). These parameters were within the acceptable range for nanomedicine formulations

315

Interestingly, the DLSLN and EESLN were the highest for SLNS1, drastically dropped for SLN2 and

316

gradually increased again for SLNS3 and SLNS4. A reasonable explanation was as follows. It was

317

established that a higher surfactant content in the formulation led to higher DL and EE 55, which

318

might be the result of the drug incorporation in the surfactant-based micelle. Accordingly, SLNS1,

319

which had the highest Tween 80 content, exhibited the highest DLSLN and EESLN. As the content of

320

Tween 80 decreased in SLNS2, the DLSLN and EESLN also decreased. The further increases in the

321

PP content in SLNS2 and SLNS3 provided more accommodation for BUD/P4, and thus led to the

322

increase in DLSLN and EESLN 56. Noticeably, there were no significant differences between DLSLN

323

and DLSLNS (p > 0.05 if DLSLNS was converted into w/w% through ρ values) or EESLN and EESLNS

324

(p > 0.05). It was speculated that almost all of the BUD/P4 was entrapped in the nanoparticles,

325

and very little was distributed in the aqueous phase.

326

In addition, the dynamic viscosity of SLNS formulations was determined. Overall, all

327

formulations exhibited non-Newtonian behavior

328

SLNS3 and SLN4 were within 1.6×10-3~1.4×10-3 Pa·s, 2.5×10-3~1.6×10-3 Pa·s, 2.9×10-2~0.9×10-2

329

Pa·s and 3.1×100~0.4×100 Pa·s, respectively, and those of b-SLNS, BUD-SLNS and P4-SLNS

330

(with the same particle size) were similar to each other (Fig. 3 A~C). These viscosity values

331

suggested that SLNS formulations were suitable for aerosolization

332

inferred that the viscosity was proportional to the lipid content in the formulation, which was

333

consistent with the previous work 59.

334

57

54

.

. The dynamic viscosities of SLNS1, SLNS2,


16


58

. Furthermore, it could be


335

TEM micrographs of the SLNS formulations are shown in Fig. 4. All micrographs demonstrated

336

the spherical or spheroidal morphology of the SLNS formulations, and the particle sizes were

337

consistent with those determined by DLS.

338


339

These results demonstrated that SLNS formulations with different particle sizes and acceptable

340

physicochemical attributes could be reproducibly fabricated. These formulations could serve as

341

the model nanoparticles for subsequent in vitro and in vivo validation of the HM device.

342

3.2 HM device validation

343

3.2.1 In vitro studies

344

The SLNS formulations (b-SLNS, BUD-SLNS and P4-SLNS) were aerosolized using HM in vitro.

345

The primary question was whether the loaded samples could be effectively and reproducibly

346

aerosolized in vitro. Consequently, the in vitro ER was evaluated (Fig. 3D). The results indicated

347

that the aerosolization volume 150 µL (ER150) exhibited the lowest ER (p < 0.05). This might be

348

attributed to a defect in the device that resulted in a failure to aerosolize the loaded sample. In

349

contrast, the 50 µL (ER50) and 100 µL (ER100) aerosolization volumes resulted in high ER values (>

350

90%). There were no significant differences between ER50 and ER100 for any of the formulations

351

(p > 0.05). The 100 µL aerosolization volume provided a more reproducible aerosolization effect

352

as demonstrated by the lower deviation of ER100. Hence, 100 µL was considered to be the optimal

353

aerosolization volume and was used in the subsequent investigation.

354

As the criterion for the use of a nebulization or metered-dose inhalation device 60, a qualified ETA

355

device should not impact the major physicochemical characteristics of the loaded samples. The

17


Page 18 of 42

Page 19 of 42 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


356

influence of in vitro aerosolization on particle size distribution, ZP, DL and EE of SLNS

357

formulations was explored, and the results are depicted in Fig. 5. For clarity, a graphical summary

358

of the related data interpretation is shown in Fig. S2.

359

No pronounced differences between the values before and after in vitro aerosolization were

360

observed in particle size, PdI, ZP, DLSLN, EESLN, DLSLNS and EESLNS (p > 0.05). However, the

361

deviation of PdI was relatively high (a possible explanation was stated in Section 3.1.2). The PdI

362

values after in vitro aerosolization remained below 0.25. Notably, the unchanged DL and EE

363

indicated that the nanostructure of SLNS was not damaged, and no drug leakage occurred. These

364

results demonstrated that in vitro aerosolization had no pronounced effect on physicochemical

365

characteristics of the SLNS formulations.

366


367

Apart from the abovementioned physicochemical characteristics, the influence of in vitro

368

aerosolization on the fluorescence spectrum of P4-SLNS still needed to be determined. First, the

369

fluorescence spectrum of raw P4 was determined and is displayed in Fig. S3. The maximum

370

excitation wavelength (λex) and maximum emission wavelength (λem) were 651 nm and 673 nm,

371

respectively. The fluorescence spectra of P4-SLNS before and after in vitro aerosolization were

372

then obtained at λex = 651 nm as shown in Fig. 6 A1~A5. No marked change in the fluorescence

373

spectra was observed. The peak fluorescence intensity of each spectrum was recorded, and the in

374

vitro aerosolization process was shown to cause less than 5% loss of peak intensity in all

375

formulations (Fig. 6 B1~B5), which was considered negligible. It should be pointed out that the

376

loss of peak intensity was the lowest for the P4 solution. This was because the extraction of P4

377

from P4-SLNS undermined the fluorescence intensity to some extent. The in vitro aerosolization

18



378

did not influence the fluorescence spectrum of P4-SLNS.

379


380

Sample residue is an important consideration for pulmonary drug delivery devices 61. Specifically,

381

a device could not be qualified if a considerable amount of sample residues was detected. In this

382

study, the sample residues from BUD-SLNS and P4-SLNS were assessed. b-SLNS was excluded

383

due to the difficulty in assaying PP by chromatography. The results showed that all residual

384

amounts were less than 3 µL/100 µL, which indicated low drug wastage and guaranteed dose

385

accuracy. Furthermore, the residual amount increased from SLNS1 to SLNS4 (p < 0.05, Fig. 7).

386

This might be associated with the increasing viscosity from SLNS1 (~10-3 Pa·s) to SLNS4 (~100

387

Pa·s). It is commonly believed that liquid samples with higher viscosity would be more “sticky”

388

on the devices inner wall and more difficult to aerosolize, resulting in a greater residue 62. Thus,

389

the loaded sample should not be highly viscous (e.g., 101 Pa·s or higher) to minimize the residue.

390

There was no detectable residue in the device after a single wash with 10 mL ethanol absolute, as

391

seen from Fig. 7, which could serve as a guide for the device rinsing during the application.

392


393

3.2.1 In vivo studies

394

Because encouraging results were obtained in the in vitro aerosolization studies, the in vivo

395

validation was then performed. The pulmonary deposition of BUD was explored after ETA of the

396

BUD-SLNS formulations. As shown in Tab. 2, the BUD pulmonary concentration was

397

proportional to the DL and EE. However, both the total recovery and emission recovery were

398

similar for BUD-SLNS1~BUD-SLNS4 (p > 0.05), which indicated a formulation-independent

19


Page 20 of 42

Page 21 of 42 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


399

ETA performance of the device. The total recovery and emission recovery were satisfactory (c.a.

400

80%), and the deviation was relatively low, which was favorable for ETA.

401

(Please insert Tab. 2 here.)

402

Encapsulation of fluorescence probes into a nanoparticulate vector enabled the visualization of the

403

in vivo deposition of the administered formulations

404

loaded in SLNS and delivered to SD rats via ETA, and typical images are shown in Fig. 8. The

405

maximum autofluorescence signal of SD rats was approximately 3400 CPS (Fig. S4). Hence, the

406

threshold of fluorescence signal in images after ETA was set to 4000 CPS to avoid the interference

407

of autofluorescence. As shown in Fig. 8 A1~A4, the autofluorescence was successfully eliminated.

408

It was unambiguously shown that the fluorescence signal, or the delivered P4-SLNS, was located

409

in the pulmonary region (Fig. 8 B1~B4). Additionally, the overall fluorescence signal and

410

area-normalized signal roughly correlated with the DL of SLNS (Fig. 8 C), which was consistent

411

with the results for the recovery of the BUD deposited in the pulmonary region. The fluorescence

412

signal of the excised lungs is shown in Fig. S5 and paralleled the in vivo bioimaging results. The

413

distribution of the fluorescence signal tended to locate in the central-lobular part of lungs, or

414

parallel along the trachea. In summary, the P4-SLNS formulations were successfully delivered to

415

the pulmonary region through ETA.

63

. In this paper, an ACQ probe (P4) was

416


417

The HM device was validated in vitro and in vivo, and the results confirmed that it was a qualified

418

device for ETA. The device could be utilized in further studies for noninvasive pulmonary

419

delivery of nanoparticle suspensions. In the near future, the authors plan to investigate the in vivo

420

fate

64, 65

of the inhaled SLNS with the assistance of this device, including the degradation,

20



421

adsorption, transportation and elimination.

422

4 Conclusion

423

In this study, SLNS formulations with different particle sizes were prepared and selected as the

424

model nanoparticle suspensions for the validation of the HM device, and satisfactory

425

reproducibility was demonstrated by the results of physicochemical characterization. The ER100

426

values for all of the formulations were approximately 90%, suggesting an effective in vitro

427

aerosolization performance. The physicochemical and fluorescence properties of SLNS were not

428

influenced by the in vitro aerosolization process. A negligible amount, ~3 µL/100 µL, of residue

429

was determined after aerosolization, which would not affect the dose accuracy. Dose recovery

430

from the lung was acceptable for the in vivo ETA administration, and bioimaging confirmed the

431

deposition of the inhaled SLNS in the pulmonary region. These results indicated that HM was a

432

qualified and promising tool for ETA in animals, which would pave the way for future laboratory

433

investigation of the pulmonary delivery of nanoparticle suspensions.

434

5 Acknowledgement

435

The authors would like to acknowledge the project grants from National Science Foundation of

436

China, under Grand No. 81703431 and 81673375, from 111 project under Grant No. B16047 and

437

from the Natural Science Fund Project of Guangdong Province under Grant No.

438

2016A030312013.

21


Page 22 of 42

Page 23 of 42 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


439

6 Conflict of interest

440

The authors stated that there were no conflicts of interest during the preparation of this manuscript.

441

Particularly, this work did not receive any financial support from Huironghe Technology Co., Ltd.

442

(Beijing, China), the supplier of HM device.

443

7 Author contributions

444

Zhengwei Huang wrote the manuscript and conducted the experiments. Ying Huang designed the

445

study and revised the manuscript. Cheng Ma, Xuejuan Zhang, Ling Lin and Ziyu Zhao helped to

446

perform the experiments. Xin Pan and Chuanbin Wu designed and supervised the study and

447

proof-read the manuscript.

448

8 Supporting information

449

Supporting information. Supplementary information, figures and tables associated with:

450

determination of DLSLN, EESLN, DLSLNS and EESLNS; gelation phenomenon of SLNS formulations;

451

Graphical interpretation of the influence of aerosolization on various properties; validation of

452

HPLC-UV methods for pulmonary BUD concentration quantification; fluorescence spectra of P4;

453

in vivo bioimaging of the SD rats before administration; ex vivo bioimaging.

454

455

9 References

456 457 458

1.

Selby, L. I.; Cortez-Jugo, C. M.; Such, G. K.; Johnston, A. P. R.

Nanoescapology: progress toward

understanding the endosomal escape of polymeric nanoparticles. Wires Nanomed Nanobi 2017, 9, (5). 2.

Costa-Gouveia, J.; Ainsa, J. A.; Brodin, P.; Lucia, A.

How can nanoparticles contribute to

22



459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502

Page 24 of 42

antituberculosis therapy? Drug Discov Today 2017, 22, (3), 600-607. 3.

Makled, S.; Nafee, N.; Boraie, N.

Nebulized solid lipid nanoparticles for the potential treatment

of pulmonary hypertension via targeted delivery of phosphodiesterase-5-inhibitor. International Journal of Pharmaceutics 2017, 517, (1-2), 312-321. 4.

Rosiere, R.; Van Woensel, M.; Gelbcke, M.; Mathieu, V.; Hecq, J.; Mathivet, T.; Vermeersch, M.;

Van Antwerpen, P.; Amighi, K.; Wauthoz, N.

New Folate-Grafted Chitosan Derivative To Improve

Delivery of Paclitaxel-Loaded Solid Lipid Nanoparticles for Lung Tumor Therapy by Inhalation. Molecular Pharmaceutics 2018, 15, (3), 899-910. 5.

Porsio, B.; Craparo, E. F.; Mauro, N.; Giammona, G.; Cavallaro, G.

Mucus and Cell-Penetrating

Nanoparticles Embedded in Nano-into-Micro Formulations for Pulmonary Delivery of Ivacaftor in Patients with Cystic Fibrosis. ACS applied materials & interfaces 2018, 10, (1), 165-181. 6.

Leng, D.; Thanki, K.; Fattal, E.; Foged, C.; Yang, M.

Engineering of budesonide-loaded

lipid-polymer hybrid nanoparticles using a quality-by-design approach. International Journal of Pharmaceutics 2017. 7.

Michen, B.; Geers, C.; Vanhecke, D.; Endes, C.; Rothen-Rutishauser, B.; Balog, S.; Petri-Fink, A.

Avoiding drying-artifacts in transmission electron microscopy: Characterizing the size and colloidal state of nanoparticles. Sci Rep-Uk 2015, 5. 8.

Kaur, M.; Malik, B.; Garg, T.; Rath, G.; Goyal, A. K.

Development and characterization of guar

gum nanoparticles for oral immunization against tuberculosis. Drug delivery 2015, 22, (3), 328-334. 9.

Haque, S.; Whittaker, M.; McIntosh, M. P.; Pouton, C. W.; Phipps, S.; Kaminskas, L. M.

A

comparison of the lung clearance kinetics of solid lipid nanoparticles and liposomes by following the 3H-labelled structural lipids after pulmonary delivery in rats. European Journal of Pharmaceutics and Biopharmaceutics 2018, 125, 1-12. 10. Ji, P.; Yu, T.; Liu, Y.; Jiang, J.; Xu, J.; Zhao, Y.; Hao, Y. N.; Qiu, Y.; Zhao, W. M.; Wu, C. Naringenin-loaded solid lipid nanoparticles: preparation, controlled delivery, cellular uptake, and pulmonary pharmacokinetics. Drug Des Dev Ther 2016, 10, 911-925. 11. Molina, R. M.; Konduru, N. V.; Hirano, H.; Donaghey, T. C.; Adamo, B.; Laurenzi, B.; Pyrgiotakis, G.; Brain, J. D.

Pulmonary distribution of nanoceria: comparison of intratracheal, microspray instillation

and dry powder insufflation. Inhalation Toxicology 2016, 28, (12), 550-560. 12. Duret, C.; Wauthoz, N.; Merlos, R.; Goole, J.; Maris, C.; Roland, I.; Sebti, T.; Vanderbist, F.; Amighi, K.

In vitro and in vivo evaluation of a dry powder endotracheal insufflator device for use in

dose-dependent preclinical studies in mice. European Journal Of Pharmaceutics And Biopharmaceutics 2012, 81, (3), 627-634. 13. Graczyk, H.; Bryan, L. C.; Lewinski, N.; Suarez, G.; Coullerez, G.; Bowen, P.; Riediker, M. Physicochemical Characterization of Nebulized Superparamagnetic Iron Oxide Nanoparticles (SPIONs). J Aerosol Med Pulm D 2015, 28, (1), 43-51. 14. Woods, A.; Patel, A.; Spina, D.; Riffo-Vasquez, Y.; Babin-Morgan, A.; de Rosales, R. T. M.; Sunassee, K.; Clark, S.; Collins, H.; Bruce, K.; Dailey, L. A.; Forbes, B.

In vivo biocompatibility, clearance, and

biodistribution of albumin vehicles for pulmonary drug delivery. J Control Release 2015, 210, 1-9. 15. Stocke, N. A.; Arnold, S. M.; Hilt, J. Z.

Responsive hydrogel nanoparticles for pulmonary delivery.

J Drug Deliv Sci Tec 2015, 29, 143-151. 16. Lipka, J.; Semmler-Behnke, M.; Wenk, A.; Burkhardt, J.; Aigner, A.; Kreyling, W.

Biokinetic

studies of non-complexed siRNA versus nano-sized PEI F25-LMW/siRNA polyplexes following intratracheal instillation into mice. International Journal of Pharmaceutics 2016, 500, (1-2), 227-235.

23


Page 25 of 42 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


503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546

17. Sullivan, B. P.; El-Gendy, N.; Kuehl, C.; Berkland, C.

Pulmonary Delivery of Vancomycin Dry

Powder Aerosol to Intubated Rabbits. Molecular Pharmaceutics 2015, 12, (8), 2665-2674. 18. Bollu, V.; Clark, R. S.; Carlton, R.; Meyer, K. L.

Economic Impact of Avoidable Drug Wastage in

Patients Admitted to the Hospital for an Acute Copd Exacerbation. Value Health 2015, 18, (3), A172-A172. 19. Kinaret, P.; Ilves, M.; Fortino, V.; Rydman, E.; Karisola, P.; Lahde, A.; Koivisto, J.; Jokiniemi, J.; Wolff, H.; Savolainen, K.; Greco, D.; Alenius, H.

Inhalation and Oropharyngeal Aspiration Exposure to

Rod-Like Carbon Nanotubes Induce Similar Airway Inflammation and Biological Responses in Mouse Lungs. ACS nano 2017, 11, (1), 291-303. 20. Frakking, T. T.; Chang, A. B.; O'Grady, K. A. F.; David, M.; Weir, K. A.

Reliability for detecting

oropharyngeal aspiration in children using cervical auscultation. Int J Speech-Lang Pa 2017, 19, (6), 569-577. 21. Small, C. J.; Gillespie, M.

Pharmacokinetics of Beclomethasone Dipropionate Delivered by

Breath-Actuated Inhaler and Metered-Dose Inhaler in Healthy Subjects. J Aerosol Med Pulm D 2018, 31, (3), 182-190. 22. Scichilone, N.; Benfante, A.; Bocchino, M.; Braido, F.; Paggiaro, P.; Papi, A.; Santus, P.; Sanduzzi, A. Which factors affect the choice Of the inhaler in chronic obstructive respiratory diseases? (vol 31C, pg 63, 2015). Pulm Pharmacol Ther 2015, 35, 152-152. 23. Ceelen, J. J. M.; Schols, A. M. W. J.; Thielen, N. G. M.; Haegens, A.; Gray, D. A.; Kelders, M. C. J. M.; de Theije, C. C.; Langen, R. C. J.

Pulmonary inflammation-induced loss and subsequent recovery of

skeletal muscle mass require functional poly-ubiquitin conjugation. Resp Res 2018, 19. 24. Dhanani, J. A.; Tang, P.; Wallis, S. C.; Parker, S. L.; Pandey, P.; Fraser, J. F.; Cohen, J.; Barnett, A.; Roberts, J. R.; Chan, H. K.

Characterisation of 40 mg/ml and 100 mg/ml tobramycin formulations for

aerosol therapy with adult mechanical ventilation. Pulm Pharmacol Ther 2018, 50, 93-99. 25. Bivas-Benita, M.; Zwier, R.; Junginger, H. E.; Borchard, G.

Non-invasive pulmonary aerosol

delivery in mice by the endotracheal route. European Journal of Pharmaceutics and Biopharmaceutics 2005, 61, (3), 214-218. 26. de Boer, A. H.; Hagedoorn, P.; Woolhouse, R.; Wynn, E.

Computational fluid dynamics (CFD)

assisted performance evaluation of the Twincer (TM) disposable high-dose dry powder inhaler. Journal of Pharmacy and Pharmacology 2012, 64, (9), 1316-1325. 27. Weber, S.; Zimmer, A.; Pardeike, J.

Solid Lipid Nanoparticles (SLN) and Nanostructured Lipid

Carriers (NLC) for pulmonary application: A review of the state of the art. European Journal of Pharmaceutics and Biopharmaceutics 2014, 86, (1), 7-22. 28. Ahmad, E.; Feng, Y. H.; Qi, J. P.; Fan, W. F.; Ma, Y. H.; He, H. S.; Xia, F.; Dong, X. C.; Zhao, W. L.; Lu, Y.; Wu, W.

Evidence of nose-to-brain delivery of nanoemulsions: cargoes but not vehicles. Nanoscale

2017, 9, (3), 1174-1183. 29. Xie, Y. K.; Shi, B. K.; Xia, F.; Qi, J. P.; Dong, X. C.; Zhao, W. L.; Li, T. L.; Wu, W.; Lu, Y.

Epithelia

transmembrane transport of orally administered ultrafine drug particles evidenced by environment sensitive fluorophores in cellular and animal studies. J Control Release 2018, 270, 65-75. 30. Xia, F.; Fan, W. F.; Jiang, S. F.; Ma, Y. H.; Lu, Y.; Qi, J. P.; Ahmad, E.; Dong, X. C.; Zhao, W. L.; Wu, W. Size-Dependent Translocation of Nanoemulsions via Oral Delivery. ACS applied materials & interfaces 2017, 9, (26), 21660-21672. 31. Zhao, W. L.; Carreira, E. M.

Conformationally restricted aza-bodipy: A highly fluorescent, stable,

near-infrared-absorbing dye. Angew Chem Int Edit 2005, 44, (11), 1677-1679.

24



547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574 575 576 577 578 579 580 581 582 583 584 585 586 587 588 589 590

Page 26 of 42

32. Hu, X. W.; Zhang, J.; Yu, Z.; Xie, Y. C.; He, H. S.; Qi, J. P.; Dong, X. C.; Lu, Y.; Zhao, W. L.; Wu, W. Environment-responsive aza-BODIPY dyes quenching in water as potential probes to visualize the in vivo fate of lipid-based nanocarriers. Nanomed-Nanotechnol 2015, 11, (8), 1939-1948. 33. Hu, X. W.; Fan, W. F.; Yu, Z.; Lu, Y.; Qi, J. P.; Zhang, J.; Dong, X. C.; Zhao, W. L.; Wu, W.

Evidence

does not support absorption of intact solid lipid nanoparticles via oral delivery. Nanoscale 2016, 8, (13), 7024-7035. 34. Wang, Y.; Zhu, L. Y.; Dong, Z.; Xie, S. Y.; Chen, X. J.; Lu, M. M.; Wang, X. F.; Li, X. H.; Zhou, W. Z. Preparation and stability study of norfloxacin-loaded solid lipid nanoparticle suspensions. Colloid Surface B 2012, 98, 105-111. 35. Chattopadhyay, P.; Shekunov, B. Y.; Yim, D.; Cipolla, D.; Boyd, B.; Farr, S.

Production of solid lipid

nanoparticle suspensions using supercritical fluid extraction of emulsions (SFEE) for pulmonary delivery using the AERx system. Advanced Drug Delivery Reviews 2007, 59, (6), 444-453. 36. Cipolla, D.; Gonda, I.

Inhaled nicotine replacement therapy. Asian J Pharm Sci 2015, 10, (6),

472-480. 37. Yasir, M.; Sara, U. V.

Solid lipid nanoparticles for nose to brain delivery of haloperidol: in vitro

drug release and pharmacokinetics evaluation. Acta Pharm Sin B 2014, 4, (6), 454-63. 38. Gupta, S.; Kesarla, R.; Chotai, N.; Misra, A.; Omri, A.

Systematic Approach for the Formulation

and Optimization of Solid Lipid Nanoparticles of Efavirenz by High Pressure Homogenization Using Design of Experiments for Brain Targeting and Enhanced Bioavailability. BioMed research international 2017. 39. Zhao, C.; Zhang, J.; Hu, H.; Qiao, M.; Chen, D.; Zhao, X.; Yang, C.

Design of lactoferrin modified

lipid nano-carriers for efficient brain-targeted delivery of nimodipine. Materials Science and Engineering: C 2018. 40. Beg, S.; Saini, S.; Bandopadhyay, S.; Katare, O. P.; Singh, B.

QbD-driven development and

evaluation of nanostructured lipid carriers (NLCs) of Olmesartan medoxomil employing multivariate statistical techniques. Drug Dev Ind Pharm 2018, 44, (3), 407-420. 41. Chantaburanan, T.; Teeranachaideekul, V.; Chantasart, D.; Jintapattanakit, A.; Junyaprasert, V. B. Effect of binary solid lipid matrix of wax and triglyceride on lipid crystallinity, drug-lipid interaction and drug release of ibuprofen-loaded solid lipid nanoparticles (SLN) for dermal delivery. J Colloid Interf Sci 2017, 504, 247-256. 42. Sarmento, B.; Martins, S.; Ferreira, D.; Souto, E. B.

Oral insulin delivery by means of solid lipid

nanoparticles. Int J Nanomed 2007, 2, (4), 743-749. 43. Jenning, V.; Gohla, S.

Comparison of wax and glyceride solid lipid nanoparticles (SLN (R)).

International Journal of Pharmaceutics 2000, 196, (2), 219-222. 44. Mehnert, W.; Mader, K.

Solid lipid nanoparticles Production, characterization and applications.

Advanced Drug Delivery Reviews 2012, 64, 83-101. 45. Dawoud, M. Z.; Nasr, M.

Comparison of drug release from liquid crystalline monoolein

dispersions and solid lipid nanoparticles using a flow cytometric technique. Acta Pharmaceutica Sinica B 2016, 6, (2), 163-169. 46. Pandita, D.; Kumar, S.; Poonia, N.; Lather, V.

Solid lipid nanoparticles enhance oral

bioavailability of resveratrol, a natural polyphenol. Food Res Int 2014, 62, 1165-1174. 47. Akbari, J.; Saeedi, M.; Morteza-Semnani, K.; Rostamkalaei, S. S.; Asadi, M.; Asare-Addo, K.; Nokhodchi, A.

The design of naproxen solid lipid nanoparticles to target skin layers. Colloid Surface B

2016, 145, 626-633.

25


Page 27 of 42 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


591 592 593 594 595 596 597 598 599 600 601 602 603 604 605 606 607 608 609 610 611 612 613 614 615 616 617 618 619 620 621 622 623 624 625 626 627 628 629 630 631 632 633 634

48. Gaur, P. K.; Mishra, S.; Bajpai, M.; Mishra, A.

Enhanced Oral Bioavailability of Efavirenz by Solid

Lipid Nanoparticles: In Vitro Drug Release and Pharmacokinetics Studies. BioMed research international 2014. 49. Bhattacharjee, S.

DLS and zeta potential – What they are and what they are not? J Control

Release 2016, 235, 337-351. 50. Luo, Y. C.; Teng, Z.; Li, Y.; Wang, Q.

Solid lipid nanoparticles for oral drug delivery: Chitosan

coating improves stability, controlled delivery, mucoadhesion and cellular uptake. Carbohydrate polymers 2015, 122, 221-229. 51. Chetoni, P.; Burgalassi, S.; Monti, D.; Tampucci, S.; Tullio, V.; Cuffini, A. M.; Muntoni, E.; Spagnolo, R.; Zara, G. P.; Cavalli, R. delivery:

Solid lipid nanoparticles as promising tool for intraocular tobramycin

Pharmacokinetic

studies

on

rabbits.

European

Journal

of

Pharmaceutics

and

Biopharmaceutics 2016, 109, 214-223. 52. Makwana, V.; Jain, R.; Patel, K.; Nivsarkar, M.; Joshi, A.

Solid lipid nanoparticles (SLN) of

Efavirenz as lymph targeting drug delivery system: Elucidation of mechanism of uptake using chylomicron flow blocking approach. International Journal of Pharmaceutics 2015, 495, (1), 439-446. 53. Thakkar, A.; Chenreddy, S.; Thio, A.; Khamas, W.; Wang, J.; Prabhu, S.

Preclinical systemic

toxicity evaluation of chitosan-solid lipid nanoparticle-encapsulated aspirin and curcumin in combination with free sulforaphane in BALB/c mice. Int J Nanomed 2016, 11, 3265-3276. 54. Baek, J. S.; Cho, C. W.

Controlled release and reversal of multidrug resistance by

co-encapsulation of paclitaxel and verapamil in solid lipid nanoparticles. International Journal of Pharmaceutics 2015, 478, (2), 617-624. 55. Das, S.; Ng, W. K.; Tan, R. B. H. nanoparticles

(SLNs):

Are nanostructured lipid carriers (NLCs) better than solid lipid

Development,

characterizations

and

comparative

evaluations

of

clotrimazole-loaded SLNs and NLCs? Eur J Pharm Sci 2012, 47, (1), 139-151. 56. Jose, S.; Anju, S. S.; Cinu, T. A.; Aleykutty, N. A.; Thomas, S.; Souto, E. B.

In vivo

pharmacokinetics and biodistribution of resveratrol-loaded solid lipid nanoparticles for brain delivery. International Journal of Pharmaceutics 2014, 474, (1-2), 6-13. 57. Dorraj, G.; Moghimi, H. R.

Preparation of SLN-containing Thermoresponsive In-situ Forming Gel

as a Controlled Nanoparticle Delivery System and Investigating its Rheological, Thermal and Erosion Behavior. Iran J Pharm Res 2015, 14, (2), 347-358. 58. Fitzgerald, C.; Hosny, N. A.; Tong, H.; Seville, P. C.; Gallimore, P. J.; Davidson, N. M.; Athanasiadis, A.; Botchway, S. W.; Ward, A. D.; Kalberer, M.; Kuimova, M. K.; Pope, F. D.

Fluorescence lifetime

imaging of optically levitated aerosol: a technique to quantitatively map the viscosity of suspended aerosol particles. Phys Chem Chem Phys 2016, 18, (31), 21710-21719. 59. Wolf,

M.;

Klang,

V.;

Halper,

M.;

Stix,

C.;

Heuser,

T.;

Kotisch,

H.;

Valenta, C.

Monoacyl-phospatidylcholine nanostructured lipid carriers: Influence of lipid and surfactant content on in vitro skin permeation of flufenamic acid and fluconazole. J Drug Deliv Sci Tec 2017, 41, 419-430. 60. Huang, Z. W.; Wu, H.; Yang, B. B.; Chen, L. K.; Huang, Y.; Quan, G. L.; Zhu, C. N.; Li, X.; Pan, X.; Wu, C. B.

Anhydrous reverse micelle nanoparticles: new strategy to overcome sedimentation instability

of peptide-containing pressurized metered-dose inhalers. Drug delivery 2017, 24, (1), 527-538. 61. Toennes, S. W.; Geraths, A.; Pogoda, W.; Paulke, A.; Wunder, C.; Theunissen, E. L.; Ramaekers, J. G. Pharmacokinetic properties of the synthetic cannabinoid JWH-018 and of its metabolites in serum after inhalation. J. Pharm. Biomed. Anal. 2017, 140, 215-222. 62. Castor, J. M. R.; Portugal, L.; Ferrer, L.; Hinojosa-Reyes, L.; Guzman-Mar, J. L.; Hernandez-Ramirez,

26



635 636 637 638 639 640 641 642 643 644 645

A.; Cerda, V.

646

10 Abbreviations

Page 28 of 42

An evaluation of the bioaccessibility of arsenic in corn and rice samples based on cloud

point extraction and hydride generation coupled to atomic fluorescence spectrometry. Food Chem 2016, 204, 475-482. 63. Wolfbeis, O. S.

An overview of nanoparticles commonly used in fluorescent bioimaging.

Chemical Society reviews 2015, 44, (14), 4743-4768. 64. He, H. S.; Zhang, J.; Xie, Y. C.; Lu, Y.; Qi, J. P.; Ahmad, E.; Dong, X. C.; Zhao, W. L.; Wu, W. Bioimaging of Intravenous Polymeric Micelles Based on Discrimination of Integral Particles Using an Environment-Responsive Probe. Molecular Pharmaceutics 2016, 13, (11), 4013-4019. 65. He, H.; Jiang, S.; Xie, Y.; Lu, Y.; Qi, J.; Dong, X.; Zhao, W.; Yin, Z.; Wu, W.

Reassessment of long

circulation via monitoring of integral polymeric nanoparticles justifies a more accurate understanding. Nanoscale Horizons 2018.

ACQ b-SLNS1~b-SLNS4 BUD BUD-SLNS1~BUD-SLNS4 DCM DL DLSLN DLSLNS EE EESLN EESLNS ER ETA HM P4 P4-SLNS1~P4-SLNS4 PdI PP SD rats SLNS TAA VWD ZP

Aggregation-caused quenching Blank solid lipid nanoparticle suspension formulation 1~4 Budesonide Budesonide-loaded solid lipid nanoparticle suspension formulation 1~4 Dichloromethane Drug loading DL of the suspending nanoparticles DL of the entire SLNS formulations Encapsulation efficiency EE of the suspending nanoparticles EE of the entire SLNS formulations Emission ratio Endotracheal aerosolization HRH MAG-4 The code of aggregation-caused quenching probe P4-loaded solid lipid nanoparticle suspension formulation 1~4 Polydispersity index Palmityl palmitate Sprague-Dawley rats Solid lipid nanoparticle suspension Triamcinolone acetonide Variable wavelength detector Zeta-potential

647 648

27


Page 29 of 42 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


649

Table

650

Tab. 1 Formulation composition and processing parameters for SLNS formulations. PP

Tween 80

Tween 80

Homogenization

Homogenization

amount

content

volume

temperature

pressure

(g)

(%, w/v)

(mL)

(°C)

(bar)

b-SLNS1

1.5

2.0

50

70

1250

20

b-SLNS2

3.5

0.5

50

70

750

20

b-SLNS3

7.5

0.5

50

70

750

35

b-SLNS4

9.5

0.5

50

80

750

55

Homogenization Formulationa

cycle

651

a

652

b-SLNS1~b-SLNS4.

BUD-SLNS and P4-SLNS were produced by adding BUD or P4 stock solution into

653

28



654

Page 30 of 42

Tab. 2 In vivo pulmonary deposition on SD rats (n = 5). Formulation

Pulmonary concentration (ng/glung)

Total recovery (%)

Emission recovery (%)

BUD-SLNS1

182.75 ± 6.45

77.01 ± 2.72

79.45 ± 2.80

BUD-SLNS2

76.88 ± 2.53

76.33 ± 2.51

77.76 ± 2.56

BUD-SLNS3

81.68 ± 3.95

76.51 ± 3.70

82.03 ± 3.96

BUD-SLNS4

114.47 ± 4.16

77.43 ± 2.82

82.30 ± 2.99

655 656

29


Page 31 of 42 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


657

Figure captions

658

Fig. 1 A graphical representation of the preparation procedures of SLNS formulations (A) and a

659

typical image of the HM device monotype (B). I: delivery tube; II: micronebulizer; III: sample cell;

660

IV: piston; V: accessories volume-fixer. Abbreviations: HM: HRH-MAG 4 device.

661 662

Fig. 2 Physicochemical properties including particle size, PdI, ZP, DLSLN, EESLN, DLSLNS and

663

EESLNS of SLNS formulations (n = 3). Abbreviations: PdI: polydispersity index; ZP: zeta-potential;

664

DLSLN: drug loading of the suspended nanoparticles; EESLN: encapsulation efficiency of the

665

suspended nanoparticles; DLSLNS: drug loading of the entire SLNS formulations consisting of the

666

suspended nanoparticles and the aqueous phase; EESLNS: encapsulation efficiency of the entire

667

SLNS formulations consisting of the suspended nanoparticles and the aqueous phase.

668 669

Fig. 3 Dynamic viscosity of b-SLNS1~b-SLNS4 (A), BUD-SLNS1~BUD-SLNS4 (B) and

670

P4-SLNS1~P4-SLNS4 (C), and the ER of different formulations in the in vitro aerosolization tests

671

(D). Measurements were conducted in triplicate if possible. Abbreviations: b-SLNS1~b-SLNS4:

672

blank

673

formulations 1~4; P4-SLNS1~P4-SLNS4: P4-loaded SLNS formulations 1~4.

SLNS

formulations

1~4;

BUD-SLNS1~BUD-SLNS4:

budesonide-loaded

SLNS

674 675

Fig. 4 TEM micrographs of b-SLNS1~4 (upper panel), BUD-SLNS1~4 (middle panel) and

676

P4-SLNS1~4 (lower panel). The scale bars were indicated in individual images. Abbreviations:

677

b-SLNS1~b-SLNS4:

678

budesonide-loaded

blank SLNS

SLNS formulations

formulations 1~4;

1~4;

BUD-SLNS1~BUD-SLNS4:

P4-SLNS1~P4-SLNS4:

30


P4-loaded

SLNS


679

Page 32 of 42

formulations 1~4.

680 681

Fig. 5 The influence of in vitro aerosolization on physicochemical properties of b-SLNS (A1~A3),

682

BUD-SLNS (B1~B7) and P4-SLNS (C1~C7). Within a certain panel, 1~7 represented particle size,

683

PdI, ZP, DLSLN, EESLN, DLSLNS and EESLNS, respectively. The term ‘before’ and ‘after’ meant

684

before and after the in-vitro aerosolization, respectively. Measurements were conducted in

685

triplicate.

686

BUD-SLNS1~BUD-SLNS4: budesonide-loaded SLNS formulations 1~4; P4-SLNS1~P4-SLNS4:

687

P4-loaded SLNS formulations 1~4; PdI: polydispersity index; ZP: zeta-potential; DLSLN: drug

688

loading of the suspended nanoparticles; EESLN: encapsulation efficiency of the suspended

689

nanoparticles; DLSLNS: drug loading of the entire SLNS formulations consisting of the suspended

690

nanoparticles and the aqueous phase; EESLNS: encapsulation efficiency of the entire SLNS

691

formulations consisting of the suspended nanoparticles and the aqueous phase.

Abbreviations:

b-SLNS1~b-SLNS4:

blank

SLNS

formulations

1~4;

692 693

Fig. 6 The influence of in vitro aerosolization on fluorescence spectrum of P4 solution (A1) and

694

P4-SLNS formulations (A2~A5), and B1~B5 were the corresponding peak fluorescence intensity

695

in A1~A5, respectively. The term ‘before’ and ‘after’ meant before and after the in-vitro

696

aerosolization, respectively. Abbreviations: P4-SLNS1~P4-SLNS4: P4-loaded SLNS formulations

697

1~4.

698 699

Fig. 7 Results of the sample residues assessment (n = 3). The group ‘BUD-SLNS’ and ‘P4-SLNS’

700

referred to that the device was not yet washed. The group ‘BUD-SLNS after washed’ and

31


Page 33 of 42 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


701

‘P4-SLNS after washed’ meant that the device had been washed by 10 mL of ethanol absolute

702

(circa 70°C) before extraction of residual BUD or P4, respectively. Abbreviations: BUD-SLNS:

703

budesonide-loaded SLNS formulations; P4-SLNS: P4-loaded SLNS formulations.

704 705

Fig. 8 Typical images taken in the in vivo bioimaging on SD rats. The upper panel (A1~A4)

706

depicted the signal of autofluorescence before P4-SLNS administration, and the lower panel

707

(B1~B4) illustrated the in vivo deposition of P4-SLNS1~P4-SLNS4, respectively. (C) exhibited

708

the overall fluorescence signal of each image. The scale bar of the fluorescence signal was also

709

provided.

710 711

32



Fig. 1 A graphical representation of the preparation procedures of SLNS formulations (A) and a typical image of the HM device monotype (B). I: delivery tube; II: micronebulizer; III: sample cell; IV: piston; V: accessories volume-fixer. Abbreviations: HM: HRH-MAG 4 device. 121x92mm (300 x 300 DPI)


Page 34 of 42

Page 35 of 42 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


Fig. 2 Physicochemical properties including particle size, PdI, ZP, DLSLN, EESLN, DLSLNS and EESLNS of SLNS formulations (n = 3). Abbreviations: PdI: polydispersity index; ZP: zeta-potential; DLSLN: drug loading of the suspended nanoparticles; EESLN: encapsulation efficiency of the suspended nanoparticles; DLSLNS: drug loading of the entire SLNS formulations consisting of the suspended nanoparticles and the aqueous phase; EESLNS: encapsulation efficiency of the entire SLNS formulations consisting of the suspended nanoparticles and the aqueous phase. 160x160mm (300 x 300 DPI)



Fig. 3 Dynamic viscosity of b-SLNS1~b-SLNS4 (A), BUD-SLNS1~BUD-SLNS4 (B) and P4-SLNS1~P4-SLNS4 (C), and the ER of different formulations in the in vitro aerosolization tests (D). Measurements were conducted in triplicate if possible. Abbreviations: b-SLNS1~b-SLNS4: blank SLNS formulations 1~4; BUDSLNS1~BUD-SLNS4: budesonide-loaded SLNS formulations 1~4; P4-SLNS1~P4-SLNS4: P4-loaded SLNS formulations 1~4. 99x62mm (300 x 300 DPI)


Page 36 of 42

Page 37 of 42 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


Fig. 4 TEM micrographs of b-SLNS1~4 (upper panel), BUD-SLNS1~4 (middle panel) and P4-SLNS1~4 (lower panel). The scale bars were indicated in individual images. Abbreviations: b-SLNS1~b-SLNS4: blank SLNS formulations 1~4; BUD-SLNS1~BUD-SLNS4: budesonide-loaded SLNS formulations 1~4; P4-SLNS1~P4SLNS4: P4-loaded SLNS formulations 1~4. 121x91mm (300 x 300 DPI)



Fig. 5 The influence of in vitro aerosolization on physicochemical properties of b-SLNS (A1~A3), BUD-SLNS (B1~B7) and P4-SLNS (C1~C7). Within a certain panel, 1~7 represented particle size, PdI, ZP, DLSLN, EESLN, DLSLNS and EESLNS, respectively. The term ‘before’ and ‘after’ meant before and after the in-vitro aerosolization, respectively. Measurements were conducted in triplicate. Abbreviations: b-SLNS1~b-SLNS4: blank SLNS formulations 1~4; BUD-SLNS1~BUD-SLNS4: budesonide-loaded SLNS formulations 1~4; P4SLNS1~P4-SLNS4: P4-loaded SLNS formulations 1~4; PdI: polydispersity index; ZP: zeta-potential; DLSLN: drug loading of the suspended nanoparticles; EESLN: encapsulation efficiency of the suspended nanoparticles; DLSLNS: drug loading of the entire SLNS formulations consisting of the suspended nanoparticles and the aqueous phase; EESLNS: encapsulation efficiency of the entire SLNS formulations consisting of the suspended nanoparticles and the aqueous phase. 208x272mm (300 x 300 DPI)


Page 38 of 42

Page 39 of 42 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




Fig. 6 The influence of in vitro aerosolization on fluorescence spectrum of P4 solution (A1) and P4-SLNS formulations (A2~A5), and B1~B5 were the corresponding peak fluorescence intensity in A1~A5, respectively. The term ‘before’ and ‘after’ meant before and after the in-vitro aerosolization, respectively. Abbreviations: P4-SLNS1~P4-SLNS4: P4-loaded SLNS formulations 1~4. 180x405mm (300 x 300 DPI)


Page 40 of 42

Page 41 of 42 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


Fig. 7 Results of the sample residues assessment (n = 3). The group ‘BUD-SLNS’ and ‘P4-SLNS’ referred to that the device was not yet washed. The group ‘BUD-SLNS after washed’ and ‘P4-SLNS after washed’ meant that the device had been washed by 10 mL of ethanol absolute (circa 70°C) before extraction of residual BUD or P4, respectively. Abbreviations: BUD-SLNS: budesonide-loaded SLNS formulations; P4-SLNS: P4loaded SLNS formulations. 89x99mm (300 x 300 DPI)



Fig. 8 Typical images taken in the in vivo bioimaging on SD rats. The upper panel (A1~A4) depicted the signal of autofluorescence before P4-SLNS administration, and the lower panel (B1~B4) illustrated the in vivo deposition of P4-SLNS1~P4-SLNS4, respectively. (C) exhibited the overall fluorescence signal of each image. The scale bar of the fluorescence signal was also provided. 99x62mm (300 x 300 DPI)


Page 42 of 42

Page 43 of 42 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


Table of content 337x154mm (150 x 150 DPI)


Endotracheal Aerosolization Device for Laboratory ... - ACS Publications

Recommend Documents