Rapid Recovery of Clofazimine-Loaded Nanoparticles with Long-Term

Apr 20, 2018 - ... dissolution tests were also evaluated with free Cfz powders and Lamprene as controls. ...... 2004, 30, 9– 17, DOI: 10.1081/DDC-12...
2 downloads 3 Views 13MB Size
Subscriber access provided by UNIV OF NEW ENGLAND ARMIDALE

Rapid Recovery of Clofazimine-loaded Nanoparticles with Long-term Storage Stability as Anti-Cryptosporidium Therapy Jie Feng, Yingyue Zhang, Simon A. McManus, Kurt D. Ristroph, Hoang D. Lu, Kai Gong, Claire E. White, and Robert K Prud'homme ACS Appl. Nano Mater., Just Accepted Manuscript • DOI: 10.1021/acsanm.8b00234 • Publication Date (Web): 20 Apr 2018 Downloaded from http://pubs.acs.org on April 20, 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 44 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

ACS Applied Nano Materials

1

Rapid Recovery of Clofazimine-loaded Nanoparticles with Long-

2

term Storage Stability as Anti-Cryptosporidium Therapy

3

Jie Feng1, Yingyue Zhang1, Simon A. McManus1, Kurt D. Ristroph1, Hoang D. Lu1, Kai Gong2,

4

Claire White2, 3, Robert K. Prud’homme1*

5

1. Department of Chemical and Biological Engineering, Princeton University, Princeton, New

6

Jersey 08544, United States

7

2. Department of Civil and Environmental Engineering, Princeton University, Princeton, New

8

Jersey 08544, United States

9

3. Andlinger Center for Energy and the Environment, Princeton University, Princeton, New

10

Jersey 08544, United States

11 12

Abstract:

13

While the formulation of nanoparticle suspensions has been widely applied in materials and

14

life science, the recovery of nanoparticles from such a suspension into a solid state is

15

practically important to confer long-term storage stability. However, solidification while

16

preserving the original nanoscale properties remains a formidable challenge in the

17

pharmaceutical and biomedical application of nanoparticles. Herein we combined Flash

18

NanoPrecipitation (FNP) and spray-drying as a nanofabrication platform for nanoparticle

19

formulation and recovery without compromising the dissolution kinetics of the active

20

ingredient. Clofazimine was chosen to be the representative drug, which has been recently

21

repurposed as a potential treatment for Cryptosporidiosis for global health. Clofazimine was

22

encapsulated into nanoparticles with low-cost surface coatings, hypromellose acetate

23

succinate (HPMCAS) and lecithin, which were required by the ultimate application to global *Corresponding author: [email protected] ACS Paragon Plus Environment

ACS Applied Nano Materials 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

Page 2 of 44

24

health. Spray-drying and lyophilization were utilized to produce dried powders with good

25

long-term storage stability for application in hot and humid climatic zones. Particle

26

morphology, yield efficiency, drug loading and clofazimine crystallinity in the spray-dried

27

powders were characterized. The in vitro release kinetics of spray-dried nanoparticle powders

28

were compared to the analogous dissolution profiles from standard lyophilized nanoparticle

29

samples, crystalline clofazimine powder and the commercially-available formulation

30

Lamprene®. The spray-dried powders showed a supersaturation level up to 60 times

31

equilibrium solubility and remarkably improved dissolution rates. In addition, the spray-dried

32

powders with both surface coatings showed excellent stability during aging studies with

33

elevated temperature and humidity, in view of the dissolution and release in vitro.

34

Considering oral delivery for pediatric administration, the spray-dried powders show less

35

staining effects with simulated skin than crystalline clofazimine, and may be made into mini-

36

tablets without additional excipients. These results highlight the potential of combining FNP

37

and spray-drying as a feasible and versatile platform to design and rapidly recover

38

amorphous nanoparticles in a solid dosage form, with the advantages of satisfactory long-

39

term storage stability, low cost, and easy scalability.

40

Keywords: clofazimine, nanoparticles, flash nanoprecipitation, hypromellose acetate succinate,

41

lecithin, spray-drying, storage stability,

42 43

1. Introduction

44

Oral ingestion is the most convenient and commonly employed route of drug delivery due to the

45

ease of administration, patient compliance, and flexibility in the design of the dosage form.[1]

46

Currently, up to 40% of new chemical entities discovered by the pharmaceutical industry today

47

are hydrophobic compounds in Biopharmaceutics Classification System class II, with low

ACS Paragon Plus Environment

2

Page 3 of 44 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

ACS Applied Nano Materials

48

solubility.[2, 3] Low solubility issue leads to inadequate and variable bioavailability, requiring

49

very high dosing or multiple-dose treatment to achieve the desired concentration in systemic

50

circulation and therefore the desired pharmacological response.[4, 5] Hence, a key goal in poorly-

51

soluble drug formulation is solubility enhancement, which in turn affords improved

52

bioavailability and feasible dosage administration. Nanoparticle (NP) formulations can improve

53

the bioavailability of hydrophobic drugs through two mechanisms. First, NPs can be formed via

54

rapid precipitation processes to trap drug molecules in an amorphous state. The amorphous state

55

results in solubility as much as 1000X higher that of crystalline states.[6, 7] Second, the greater

56

surface-to-volume ratio for NPs versus large drug crystals enhances dissolution kinetics.

57

However, successful NP formation overcomes only the first challenge in the path to a successful

58

oral dosage form. Equally important is the recovery of the NPs into a dry, solid form without

59

comprising the enhanced dissolution kinetics and bioavailability. Common techniques for

60

solvent removal include lyophilization, salt flocculation and spray-drying.[8] Lyophilization

61

requires extremely long processing time and its high cost generally makes it a last-resort

62

solidification process. Additionally, the increase of particle concentration and changes of the

63

solvent compositions during freezing may induce various stresses that cause particle

64

crystallization and growth. Salt flocculation, or flocculation initiated by an excipient to create a

65

filterable solid that can be tray dried, has been recently advanced as a new method of making

66

redispersible powders.[8, 9] In contrast, spray-drying is a one-step, continuous and scalable drying

67

method, and is therefore an attractive candidate for large-scale NP powder processing. The

68

solidification is achieved by mixing a heated gas with an atomized liquid of fine droplets within

69

a vessel, causing the solvent to evaporate quickly through direct contact. Spray-drying requires a

70

shorter processing time than lyophilization and ultrafiltration, with the cost of drying 30 to 50

ACS Paragon Plus Environment

3

ACS Applied Nano Materials 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

Page 4 of 44

71

times less than lyophilization.[10] Moreover, the adjustment of process parameters enables the

72

direct manipulation of various particle properties, such as powder size and morphology, which is

73

particularly useful for meeting the specific requirements for various administration routes and

74

delivery systems.[11] Another advantage of spray-drying is that it can be set up as an aseptic

75

process for Good Manufacturing Practice production, with the use of filters on the gas flow,

76

sterilization of the nozzle and chamber walls, and fast product collection.[12]

77

While spray-drying of pharmaceutical solutions to produce powders is widely practiced,[13] and

78

some studies have addressed the inclusion of NPs in the solvent phase,[14-16] little is available to

79

guide in the selection of spray-drying excipients appropriate for different NP surface stabilizers.

80

Here we report on spray-drying of preformed NPs as a means to obtain dry powders that display

81

rapid drug dissolution kinetics. We specifically focus on clofazamine (Cfz), a drug that is

82

effective against the Cryptosporidium infections that are the second major cause of infant

83

mortality in sub-Saharan Africa.[17, 18] The absorption of Cfz is dissolution-limited and solubility

84

enhancement is the focus of the NP formulation development. We previously showed that NPs

85

stabilized by zein, hypromellose acetate succinate (HPMCAS), or lecithin, enhanced the

86

dissolution of clofazmine to high levels of supersaturation following lyophilization,[19] while in

87

the current work, we particularly aim to explore the rapid recovery of drug-loaded NPs with an

88

appropriate spray-drying protocol, to maintain the fast dissolution kinetics. Furthermore, we have

89

also demonstrated the potential of the spray-dried NP powders as pediatric formulations in the

90

field of global health.

91

Flash NanoPrecipitation (FNP), a stabilizer-directed rapid precipitation process, is used to

92

produce nanometer-sized particles. In FNP, amphiphilic stabilizers and hydrophobic drugs are

93

molecularly dissolved in an organic phase and mixed rapidly with an anti-solvent stream to drive

ACS Paragon Plus Environment

4

Page 5 of 44 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

ACS Applied Nano Materials

94

controlled precipitation with tunable particle size (~50-500 nm).[20] In this study, instead of using

95

synthetic diblock copolymer, we performed FNP using Cfz and two stabilizers HPMCAS and

96

lecithin. After NP formulation, lyophilization (Lyo) and spray-drying (SD) were both optimized

97

to obtain dried NP powders, in order to produce a solid oral dosage form with high tolerance to

98

temperature and humidity. The dried powders were further tested in simulated gastric and

99

intestinal fluids for in vitro release, to investigate the effect of different surface stabilizers and

100

drying pathways on the dissolution kinetics of Cfz. Long-term stability studies were then

101

performed with the SD powders to demonstrate the resistance to degradation in harsh conditions.

102

In addition, we examined both the SD powders’ skin staining effects and compressibility into

103

mini-tablets; both of these factors are relevant to the powders’ applicability as a pediatric

104

administration in sub-Saharan Africa.

105

2. Materials and Methods

106

Materials. Cfz (Fig. 1 (a)), sucrose, trehalose, mannitol and all solvents (HPLC grade) were

107

purchased from Sigma-Aldrich (Milwaukee, WI) and used as received. AFFINISOL

108

hypromellose acetate succinate (HPMCAS, USP grade) and METHOCEL HPMC E3 (viscosity

109

of 2.4−3.6 mPa·s at 2% solution in water at 20 °C, Tg=174 °C) were a gift from Dow Chemical

110

Company (Midland, MI). L-α-lecithin was purchased from Fisher Scientific (Waltham, MA).

111

FaSSIF/FeSSIF/FaSSGF and FeSSIF-V2 powders were purchased from Biorelevant.com

112

(London, U.K.). Deionized (DI) water (18.2 MΩ·cm) was prepared by a NANOpure Diamond

113

UV ultrapure water system (Barnstead International, Dubuque, IA).

114

Nanoparticle Formulation and Characterization. NPs were created via FNP first using a

115

confined imping jet mixer (CIJ, Fig. 1 (b)) to optimize the formulations. Briefly, an organic

116

stream of either acetone (for HPMCAS) or tetrahydrofuran (THF, for lecithin) with molecularly

ACS Paragon Plus Environment

5

ACS Applied Nano Materials 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

Page 6 of 44

117

dissolved Cfz and stabilizers, was rapidly mixed against a DI water stream into the mixing

118

chamber of a CIJ in a 1:1 volume ratio. The concentrations in the organic stream were 5 mg/mL

119

Cfz and 5 mg/mL HPMCAS for the HPMCAS formulation, and 10 mg/mL Cfz and 5 mg/mL in

120

the lecithin formulation. The resulting mixed stream was collected in a quenching DI water bath

121

to drop the final organic concentration to 10 vol %. NP formulations for spray-drying were

122

produced using a multi-inlet vortex mixer (MIVM, Fig. 2 (a)). The MIVM’s four-inlet geometry

123

allows higher supersaturation during mixing than the two-inlet CIJ and bypasses the secondary

124

quenching step;[21] thus this mixer is preferred for the continuous and scalable production of NPs.

125

In the MIVM set-up, one organic stream containing Cfz and stabilizers, and three other water

126

streams were mixed together, with a volumetric flow rate of organic:water=1:3, making the final

127

organic concentration as 10 vol %. Therefore, no further quenching is necessary to reach the

128

same dilution as the CIJ formulations. All NP suspensions for the spray-drying process were

129

prepared from MIVM. For the HPMCAS formulation, the concentration in the organic stream

130

was 5 mg/mL for both Cfz and the stabilizer, with a total flow rate of 160 mL/min into MIVM.

131

For the lecithin formulation, the concentration in the organic stream was 50 mg/mL for Cfz and

132

25 mg/mL for lecithin, with a total flow rate of 120 mL/min for MIVM. Accounting for the

133

dimension of the MIVM, the effective Reynolds numbers

134

and lecithin, respectively (see SI for detailed calculation). NP diameter was determined from

135

triplicate experiments by dynamic light scattering (DLS) at 25 °C with a Zetasizer Nano-ZS

136

(Malvern Instruments, Southboro, MA), using a detection angle of 173°. DLS data were

137

processed with Malvern’s software using a distribution analysis. The average size was given

138

based on a cumulant model. The cumulant analysis is defined in International Organization for

139

Standardization (ISO) standard document 13321. On the other hand, the intensity-weighted size

[22]

ACS Paragon Plus Environment

are 11280 and 7970 for HPMCAS

6

Page 7 of 44 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

ACS Applied Nano Materials

140

distribution was obtained from a distribution analysis of the “General Purpose Mode” provided

141

by the software.

142

Nanoparticle Lyophilization. Lyophilization was carried out using a benchtop VirTis

143

Advantage (Gardiner, NY) with cryoprotectants (i.e., mannitol or HPMC E3). NP solutions (0.5

144

mL) were mixed with 0.1 mL cryoprotectant solutions at different concentrations to afford

145

various final NP:cryoprotectant mass ratios up to 1:30. The mixtures were then flash frozen by

146

fast immersion in a dry ice/acetone cooling bath (−78 °C) for 1 min with mild agitation. The

147

frozen samples were then immediately transferred to the lyophilizer with shelf temperature at

148

−20 °C under vacuum (0.366 g/mL). The desiccator was placed inside a gravity

219

convection general incubator (VWR, Radnor, PA) with a temperature control. The conditions

220

inside the desiccator were kept as 40 °C and 70% relative humidity (RH), which was

221

continuously monitored with a hygro/thermometer in the desiccator and a wireless reader outside

222

the incubator (TraceableTM, Thermo Fisher Scientific, Waltham, MA). The dissolution tests

223

described above were performed at day 0, 7, 14, month 1 and 2 to monitor the release kinetics of

224

the powders.

225

Staining Test. Polyurethane membrane (Sigma-Aldrich, St. Louis, MO) was cut into 2cm x 2cm

226

strips and adhered on the bottom of FisherbrandTM petri dishes (Fischer Scientific, Hampton,

227

NH). NP powders were suspended in DI water at concentration equivalent to Cfz at 3.33 mg/mL

ACS Paragon Plus Environment

10

Page 11 of 44 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

ACS Applied Nano Materials

228

at room temperature for 30 min. A 1 mL aliquot of the NP suspension was then applied on top of

229

the polyurethane membrane followed by incubation for 5 min. The NPs were removed via

230

pipetting and the membrane was rinsed by 1 mL DI water 3 times to remove any loosely attached

231

NPs. To quantify the Cfz staining on the membrane, the polyurethane membrane was peeled off

232

and Cfz was extracted with 2 mL of acetone. The Cfz concentration was analyzed by a UV-Vis

233

spectrophotometer at 450 nm (Evolution 300 UV-Vis, Thermo Electron, Waltham, MA).

234

Compressed Mini-tablet Test. Flat-surfaced 3 mm mini-tablets were prepared by direct

235

compression using Gamlen Tablet Press (GTP-1, Gamlen Tableting, Ltd., Nottingham, UK). The

236

die was manually filled with pre-weighed NP powders. The compression pressure corresponded

237

to a load of 100 kg.

Fig. 1. (a) Chemical formula of clofazimine; (b) schematic set-up of the protected nanoparticles made with the confined impinging (CIJ) mixer; (c) size stability of clofazimine nanoparticles with various surface coatings in 10 vol % organics (HPMCAS126, ; HPMCAS-716, ; HPMCAS-912, ; lecithin, ) 238

3. Results and discussion

239

Nanoparticle Formulations by CIJ and MIVM. HPMCAS is a cellulose ester bearing acetyl

240

and succinyl groups, which is synthesized by functionalizing HPMC with a mixture of

241

monosuccinic acid and acetic acid esters.[23] The dissolution behavior of this polymer in different

ACS Paragon Plus Environment

11

ACS Applied Nano Materials 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

Page 12 of 44

242

pH buffers may be tuned by changing the ratio of succinoyl and acetyl moieties. HPMCAS has

243

been widely used to create stable amorphous solid dispersions with poorly soluble active

244

pharmaceutical ingredients.[24, 25] Although several studies examined HPMCAS as a carrier in hot

245

melt extrusion and spray-drying, formulating NPs with HPMCAS as a surface stabilizer has

246

received little attention. In the current study, three standard commercial GMP grades of

247

HPMCAS, HPMCAS-126, 716 and 912, were investigated. They are differentiated by the ratio

248

of acetyl and succinyl substituents on the HPMC backbone.

249

Our previous work on nanoprecipitation had used synthetic diblock copolymer, where the

250

hydrophobic domain anchors on the NP surface and the hydrophilic (e.g., PEG) domain sterically

251

stabilizes the NP. It was unclear whether the randomly hydrophobic HPMCAS would create

252

stable NPs, or the hydrophobic blocks would cause bridging between NPs. Our initial hypothesis

253

was that the most hydrophobic HPMCAS would lead to the most unstable NP formulation, but

254

discrepancy was observed in the experiments. Regarding the stability of Cfz NPs prepared with

255

CIJ as shown in Fig. 1(c), HPMCAS-126 nanoparticles were the most stable formulation with no

256

size change for at least 6 h, while the size of HPMCAS-716 nanoparticles increased significantly

257

from 70 nm to around 1 µm in 3 h. With the highest acetyl substitution level, HPMCAS-126 is

258

the most hydrophobic of the three HPMCAS polymers. This cellulose derivative may interact

259

with the Cfz core more strongly than HPMCAS-716 and 912, resulting in longer nanoparticle

260

stability. Therefore, HPMCAS-126 was selected for the remainder of this study. MIVM was

261

further used to provide a large batch of NP suspensions for spray-drying. Due to different mixer

262

geometry and mixing solvent ratio, the size of HPMCAS-126 NPs increased to 150 nm from 90

263

nm in CIJ, but again remained constant for 6 h (Fig. 2 (b)).

ACS Paragon Plus Environment

12

Page 13 of 44 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

ACS Applied Nano Materials

264

Lecithin designates the group of phospholipid substances occurring in animal and plant tissues. It

265

is an essential component of cell membrane and has been widely utilized as stabilizer to form

266

nanoparticles for controlled drug delivery.[26] L-α-lecithin derived from soybean was used in the

267

current study. For lyophilization trials, lecithin NPs produced with a CIJ mixer were 175 nm in

268

diameter and maintained that size for around 3 h (Fig. 1(b)). When testing this formulation to

269

produce a SD powder, low mass yield efficiency was observed. To increase the mass yield

270

efficiency in the spray-drying, 2.5× higher concentrations of Cfz and lecithin were used in

Fig. 2. (a) Schematic set-up of the protected nanoparticles made with a multi-inlet vortex mixer (MIVM) for the spray-drying process; (b) size stability of clofazimine nanoparticles generated by MIVM with HPMCAS-126 and lecithin in 10 vol % organics (HPMCAS-126, ; lecithin, ). 271

MIVM, which greatly improved the spray-drying yield efficiency as descried below. These

272

MIVM-generated lecithin NPs had an average initial diameter of 432 nm, which grew to 540 nm

273

in 3 h and then 680 nm in 6 h (Fig. 2 (b)). Lecithin NPs have a shorter stability window

274

compared with HPMCAS NPs. Polymeric stabilizers, because of multi-valent surface attachment

275

and thicker steric layers, are often more effective than single-tailed small molecular

276

surfactants.[23] All the drying processes were performed immediately after NP formulation to

277

minimize the influence of the size growth on the in vitro release experiments.

ACS Paragon Plus Environment

13

ACS Applied Nano Materials 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

Page 14 of 44

278

Lyophilization of clofazimine nanoparticles. Lyophilization, also known as freeze-drying, is

279

generally considered as the “gold standard” for obtaining redispersible solid powders from

280

nanoparticle suspensions. However, large-scale lyophilization is prohibitively expensive for

281

drugs intended for global health. Lyophilization consists of removing solvents from a frozen

282

sample by sublimation and desorption under vacuums.[27] Stresses induced by freezing and

283

dehydration, such as the mechanical force exercised by crystallization of ice, may induce

284

irreversible aggregation of NPs. Therefore, cryoprotectants are commonly required to preserve

285

NP integrity.

286

Sucrose, trehalose, mannitol, and HPMC E3 were screened at different NPs:cryoprotectant mass

287

ratios. These cryoprotectants were selected because they rely upon a different mechanism of

288

protection. During freezing, sucrose and trehalose form glass phases with high Tg, while

289

mannitol forms crystal domains. Such a matrix of the cryoprotectant immobilizes NPs,

290

preventing their aggregation and protecting them against the mechanical stress of ice crystals.[28]

291

HPMC E3 was chosen because its cellulose backbone is compatible with that of HPMCAS, and

292

the lack of hydrophobic substitution should enable it to coat the NPs without bridging between

293

NPs. Upon reconstitution with water, considerably large particles (on the order of µm) were

294

obtained for both Cfz-loaded HPMCAS and lecithin NPs with all cryo-protectants. Additionally,

295

HPMCAS NPs with sugar cryoprotectants generated densely packed Lyo cakes, while fluffy Lyo

296

cakes were formed when using HPMC E3 with a mass ratio as low as 1:0.5. Since the high

297

porosity and open structure of such fluffy Lyo cakes increased the surface area and therefore

298

facilitated reconstitution, HPMC E3 was chosen as the favorable cryoprotectant for HPMCAS

299

NPs. Further increasing of the HPMC E3 concentrations only marginally reduced particle size

300

and polydispersity. To maximize the drug loading in final formulation while maintaining good

ACS Paragon Plus Environment

14

Page 15 of 44 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

ACS Applied Nano Materials

301

redispersibility, HPMCAS NPs with HPMC E3 cryoprotectant at a mass ratio of 1:0.5 were used

302

for the following dissolution tests. Lecithin NPs lyophilized with mannitol with a mass ratio of

303

1:3 produced a less dense Lyo cake compared with other cryoprotectants,[19] and was thus chosen

304

as the cryoprotectant for further study.

305

The difference in optimal cryoprotectants for the two NP types is related to the differences

306

between HPMCAS and lecithin as surface stabilizers. The hydrophobic substitution on

307

HPMCAS makes it interact with the hydrophobic Cfz core strongly. However, it is also

308

interactive with other HPMCAS molecules if forced into contact. For example, if the Cfz NP

309

suspension is centrifuged to pellet the NPs, the resulting pellet did not redisperse back by

310

addition of water. HPMC E3 is a less hydrophobic cellulose that is miscible with HPMCAS;

311

however, it has a relatively weak interaction with itself or HPMCAS-126. Therefore, HPMC E3

312

acts as a protective layer around the NPs that prevents direct NP contact during lyophilization,

313

enabling redispersion. In contrast, the lecithin stabilizing layer is a very thin, zwitterionic layer

314

that stabilizes by electrostatic repulsion. In this case, the mannitol sugar crystallizes around the

315

NP, resulting in a crystalline shell that prevents particle aggregation during drying.[14, 28]

ACS Paragon Plus Environment

15

ACS Applied Nano Materials 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

Page 16 of 44

Fig. 3. (a) Bright-field microscope image of the SD HPMCAS NP powders (mass ratio of NPs to HPMC E3=1:2). Inset: scanning electron microscope image of the powders; (b) size distribution of the HPMCAS NP suspension before spray-drying and the aqueous suspension after reconstituting SD powders with DI water, for different mass ratios between NPs and HPMC E3 in the spray-drying process. 316

Spray-drying of clofazimine nanoparticles. Since spray-drying is a continuous processing

317

operation involving a combination of several stages, including atomization, mixing of the spray

318

with the drying gas, evaporation, and production separation, it is important to optimize the

319

operating conditions based on the quality requirements for the final product.[29] For instance, the

320

inlet gas temperature significantly affects the physical state of the drug, residual moisture content

321

and particle morphology.[30] In addition, the excipients found most successful for lyophilization,

322

HPMC E3 and mannitol, were added into the NP suspension after FNP and prior to spray-drying .

323

These act as a coating material protecting NPs against oxidation and irreversible particle-particle

324

aggregation during drying.

325

Table 1 summarizes the characterizations of the SD powders, including the glass

326

transition/melting temperature of stabilizers, inlet gas temperature, particle diameter, loading

327

capacity (LC), mass yield efficiency (YE), residual moisture content and redispersity in water.

ACS Paragon Plus Environment

16

Page 17 of 44 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

ACS Applied Nano Materials

328

For HPMCAS NPs, the operating conditions were optimized to be: inlet temperature of 150 ºC,

329

drying gas flow rate of

330 331 332 333 334

Table 1. Characterizations of SD powders with HPMCAS and lecithin as the stabilizers, including the glass transition/melting temperature of stabilizers, inlet gas temperature of spray-drying, SD particle diameter, loading capacity, mass yield efficiency, residual moisture content and redispersity in waters.

Samples

Stabilizer Diameter Tinlet Tg or Tm (d10%/d50%/d90%) (ºC) (µm) (ºC)

HPMCAS126 NPs

Tg=120*

150

3.7/6.7/9.1

Lecithin NPs

Tm=45**

125

3.5/5.9/8.4

LC (wt %) 14.3±0.2 vs. 16.7% in formulation 17.8±0.2 vs. 16.7% in formulation

YE (wt %)

Residual Size after moisture redispersion (wt %)

45±9

3.1±0.2

250±5 nm

60±5

0.7±0.1

1.5±0.9 µm

335 336 337 338 339

300 L/h (at standard temperature and pressure), feed rate of the NP suspension of 6 mL/min, and

340

the aspirator setting of 90% (gas flow rate of 35 m3/h). Using microscopy, the volumetric

341

diameter was determined to be d10%=3.7 µm, d50%=6.7 µm and d90%=9.1 µm based on the image

342

analysis for 200 particles. The outlet filter blocked all these microparicles, and no individual NP

343

was produced and released to the air during the spray drying. YE for HPMCAS N Ps was slightly

344

lower than that of lecithin NPs since the dried powders were sticker and adhered more to the

345

drying chamber of the spray-drier, resulting in a larger mass loss. On the other hand, the higher

346

residual moisture content of SD HPMCAS powders might come from the fact that the total solid

347

concentration in the HPMCAS NP suspension was one third of that in lecithin NP suspension, so

348

the HPMCAS powders were not completely dried due to the higher water content in the

349

atomized droplets. Also, the morphology of the SD powders was observed to be shriveled,

* See https://www.dow.com/en-us/pharma/products/affinisol ** See Fig. S5 in [19]

ACS Paragon Plus Environment

17

ACS Applied Nano Materials 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

350

instead of dense spheres (Fig. 3(a)). Two characteristic times are critical in the drying process for

351

formation of such dimpled and hollow powders.[15] The first is the time required for a droplet to

352

dry, %&'( , while the second is the time required for a solute or NP to diffuse from the surface of

353

the droplet to its center, ) * /4-, where ) is the diameter of the droplet and - is the solute or NP

354

diffusion coefficient. The ratio of these two characteristic times defines a dimensionless mass

355

transport number as ./ = ) * /(4-%&'( ), which describes the relative importance of diffusion and

356

convection dynamics. For the current spray-drying condition, ./ ≈O(10) for HPMC E3 and

357

./≈O(103) for HPMCAS NPs (see SI for detailed calculations). Therefore, the slower diffusion

358

of NPs allows them to accumulate on the free surface and form a shell during drying, but since

359

the shell is further compressed by the large capillary forces of the shrinking droplet, the shell

360

would buckle or fold, showing the final wrinkled morphology as shown in Fig. 3(a).[31]

361

Importantly, when HPMCAS NPs were spray-dried along with HPMC E3 at a NP:HPMC E3

362

mass ratio greater than 1:2, the resulting powders could be redispersed to the nanoscale size (Fig.

Fig. 4. (a) Bright-field microscope image of the SD lecithin-NP powders. Inset: scanning electron microscope image of the powders. The mass ratio between lecithin NPs and mannitol is 1:3; (b) Microscope image of the aqueous suspension after reconstituting SD powders with DI water. Inset: microscope image with a larger 18 ACS Paragon Plus Environment magnification (40X).

Page 18 of 44

Page 19 of 44 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

ACS Applied Nano Materials

363

3(b)). After spray-drying, the microparticles were formed by HPMCAS NPs in a polymeric

364

matrix of HPMC E3. When redispersed with water, HPMC E3 were dissolved and HPMCAS

365

NPs were resuspended into the aqueous phase. Such an observation confirmed the high-

366

temperature spray-drying did not induce a significant degree of aggregation.

367

We note that with lyophilization, the dried powders with the same mass ratio of excipient could

368

not be readily redispersed to NPs with DI water, and formed large microparticles instead, even

369

upon sonication. For HPMCAS NPs, it was unexpected that spray-drying gave superior

370

redispersion over lyophilization.

371

For lecithin NPs, the operating conditions were optimized to be: inlet temperature of 125 ºC,

372

drying gas flow rate of 473 L/h (at standard temperature and pressure), feed rate of the NP

373

suspension of 4 mL/min, and the aspirator setting of 90% (gas flow rate of 35 m3/h). The lower

374

inlet temperature compared with HPMCAS was chosen since the degradation of L-α-lecithin

375

occurs at around 150 ºC.[19] The resulting powder’s volumetric diameter was determined to be

376

d10%=3.5 µm, d50%=5.9 µm and d90%=8.4 µm based on the image analysis for 200 particles (Fig. 4

377

(a)). Although ./≈O(103) for lecithin NPs (see SI for detailed calculations), the surface of the

378

powder was much smoother compared with the case of HPMCAS coating, indicating that

379

mannitol was able to form a shell that could resist the capillary force during fast drying without

380

buckling. In addition, it is known that mannitol added in the formulation promotes the formation

381

of spherical and smooth-surfaced microparticles.[32] Also, both the SD and Lyo lecithin powders

382

could only be redispersed to micrometer-sized colloids, even when large amounts of mannitol

383

were added as excipients (Fig. 4 (b)). Previously we had shown that mannitol could be used as a

384

spray freeze drying excipient for lecithin nanoparticles, and that the nanoparticles could be

385

redispersed from the powder.[14] These results indicate that the much faster freezing conditions

ACS Paragon Plus Environment

19

ACS Applied Nano Materials 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

Page 20 of 44

386

for spray freeze drying are required to prevent lecithin NP aggregation. However, as discussed

387

below, the level of aggregation does not compromise rapid dissolution of either lecithin

388

formulation. Compared with polymeric HPMCAS, lecithin is a small-molecular-weight molecule

389

with short hydrophobic tail and zwitterionic headgroup. Hence, it is expected that the thicker

390

adsorbed polymer layer of HPMCAS is more effective at NP stabilization than lecithin, resulting

391

in a better redispersity of SD NPs stabilized by HPMCAS than lecithin.

392

XRPD and DSC X-ray powder diffraction and calorimetric techniques have been used to

393

identify the physical state of drug molecules in a complex polymeric matrix.[33] They are useful

394

to study changes in crystallinity of the drug, which could be one of the mechanisms responsible

395

for improved dissolution.[34] With XRPD, the major diffraction peak in the signal of Cfz crystals

396

appears at 20=22.1º (Cu Kα radiation), which was in good agreement with previous studies.[35]

397

The reduction of crystallinity of drugs encapsulated in the NPs was indicated by a decrease of the

398

intensity, as well as peak broadening at 20=22.1º for Lyo and SD NPs (highlighted by the

399

dashed box in Fig. 5 (a)). In addition, DSC with Cfz crystals shows a sharp melting endotherm at

400

223.3 °C, followed by an exothermal peak due to degradation. However, melting endotherms for

401

all NP samples shifted to a lower temperature and had a broader peak width (highlighted by the

402

dashed box in Fig. 5 (b)), confirming lower crystallinity of the drug in the NPs. Hence, both

403

XRPD and DSC results indicate a reduction in the level of crystallinity for Cfz encapsulated by

404

FNP compared with raw Cfz powders. For HPMCAS coating, XRPD signal of Lyo NPs show a

405

relatively more pronounced peak at 20=22.1º compared with SD NPs, suggesting a higher

406

crystallinity in the Lyo powders. For the lecithin-coating NPs, new diffraction peaks for NPs

407

appear due to the crystallization of the excipient, mannitol, during the drying processes, which

408

mask the Cfz peaks. Although it is difficult to directly compare between the Lyo and SD lecithin

ACS Paragon Plus Environment

20

Page 21 of 44 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

ACS Applied Nano Materials

409

samples using XRPD as mannitol is known to have different crystal states,[36] the appearance of a

410

small peak at around 227 °C in the DSC signal of the SD lecithin NPs indicates a higher level of

Fig. 5. (a) X-ray powder diffraction pattern and (b) heat flow from differential scanning calorimetry for clofazimine powder and HPMCAS/lecithin NPs with different drying processes. Inset: Close-up of X-ray powder diffraction pattern for SD and Lyo HPMCAS NPs 411

crystallinity compared with Lyo lecithin NPs.

412

Dissolution Tests. Pharmaceutical solid oral dosage forms must undergo dissolution in the

413

intestinal fluids of the gastrointestinal tract before they can be absorbed and achieve the systemic

414

circulation. Therefore, dissolution is a critical part of the drug-delivery process. The 24 h

415

solubility of Cfz in FaSSGF, FaSSIF, and FeSSIF was determined to be 0.36, 6.20, and 29.60

416

µg/mL, respectively. The enhanced solubilizing capacity of intestinal fluids compared with

417

gastric fluid can be attributed to bile and pancreatic secretions and the presence of exogenous

418

lipid products.[37] We performed experiments to test Cfz dissolution kinetics in vitro for powders

419

using different stabilizers and drying processes following previously established protocol.[19]

ACS Paragon Plus Environment

21

ACS Applied Nano Materials 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

Page 22 of 44

420

Lyo or SD Cfz samples were dispersed in FaSSGF with an initial concentration of ~208× the

421

equilibrium solubility of free Cfz powder. Cfz powders (raw drug crystalline) and the

422

commercial product Lamprene® were included as control samples. Lamprene is formulated as a

423

microcrystalline suspension of Cfz in an oil-wax base in gelatin capsules. The evolution of

424

concentration of Cfz dissolved in the FaSSGF from various samples during a 30-minute

425

exposure is shown in Fig. 6, in which the supersaturation level is defined as the ratio between the

426

dissolved Cfz concentration and the solubility of Cfz crystalline. The Cfz powder only reached

427

the solubility limit of 0.36 µg/mL, while Lamprene showed a plateau at around 2.2 µg/mL (~6×

428

solubility) after 1 minute incubation. However, HPMCAS and lecithin NPs quickly achieved a

Fig. 6. (a) Dissolution kinetics and (b) supersaturation level of HPMCAS/lecithin NPs with different drying processes compared to clofazimine powder and Lamprene in FaSSGF. 429

much higher supersaturation right after dispersion, with Cfz concentration up to 57× solubility

430

for the case of SD HPMCAS NPs. The supersaturation levels for the two coatings were not

431

significantly different and were in the range of 49-57× solubility. The maximum supersaturation

432

of lecithin NPs occurred within the time resolution of the first measurement, which was 1 minute,

433

while HPMCAS NPs showed a prolonged supersaturation; the latter can be explained by a

ACS Paragon Plus Environment

22

Page 23 of 44 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

ACS Applied Nano Materials

434

previous study’s findings that HPMCAS inhibits crystallization from the supersaturated solution

435

generated by dissolution of the amorphous material.[38] Importantly, there was no significant

436

difference in the supersaturation curves between spray-drying and lyophilization in the case of

437

both HPMCAS and lecithin stabilizers, which indicates the low level of crystallinity of the

438

encapsulated drugs are preserved well even in the high-temperature spray-drying. The area under

439

the curve (AUC) of Cfz over time for SD NPs was ~1.3 times that of Lyo for both HPMCAS and

440

lecithin. Here AUC reflects the total drug exposure of Cfz in FaSSGF over the 30-minute

441

experimental period, and hence larger AUC demonstrates a higher bioavailability enhancement.

442

Next, after 30-minute incubation at 37 ºC and pH=1.6, NP/gastric fluid solution was further

443

diluted into FaSSIF and FeSSIF to simulate the fasted and feasted conditions in the intestine. The

444

release kinetics regarding the Cfz concentration at different time points are shown in Fig. 7. Here

445

the percentage of release is defined as the mass ratio between the dissolved drug and the total

446

drug put into FaSSIF and FeSSIF. All samples show a faster dissolution rate in fed state since the

447

higher lecithin and bile salt concentration helps solubilize the lipophilic drug.[39] Within 6 h, the

448

Cfz powder and Lamprene only demonstrated release of 20-30% in FaSSIF and 50-70% in

449

FeSSIF, while NP samples reached a much faster release in both simulated intestinal fluids,

450

which is highly desirable as Cryptosporidiosis infections mostly reside in the intestine.[18]

451

HPMCAS NPs have a slower release compared with lecithin NPs in these intestinal conditions.

452

In simulated gastric condition, the presence of lecithin stabilizer may have modified the form of

453

recrystallized Cfz, and consequently, the drug was more susceptible to dissolution under the

454

intestinal fluid conditions. Compared with NPs solidified by Lyo, SD NPs displayed similar fast-

455

release behavior. The faster release of SD HPMCAS NPs in FeSSIF demonstrates a lower

456

crystallinity of Cfz encapsulated in SD powders than in Lyo NPs, as confirmed by the

ACS Paragon Plus Environment

23

ACS Applied Nano Materials 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

Page 24 of 44

457

redispersity and the XRPD observation (Fig. 5 (a)). On the other hand, consistent with the DSC

458

results for Lyo and SD lecithin NPs, SD lecithin NPs show a slower dissolution rate in FaSSIF

459

due to the negative impacts of temperature elevation during spray-drying for lecithin coating; but

460

the release was still much faster than Cfz powder and Lamprene. Therefore, a recovery process

461

based on scalable and cost-effective spray-drying is a viable route to maintain the dissolution

462

properties of NPs, which is crucial to enable easy access of the therapeutics to target patients in

463

developing countries.

ACS Paragon Plus Environment

24

Page 25 of 44 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

ACS Applied Nano Materials

Fig. 7. Dissolution kinetics of HPMCAS/lecithin NPs with different drying processes compared to clofazimine powder and Lamprene in (a) FaSSIF and (b) FeSSIF. 464

Long-term Storage Stability. The long-term storage stability of dried powders is a critical

465

property for medical applications of NPs. In particular, regarding this Cfz-loaded NP formulation

466

for global health, the hot and humid climates in tropical and equatorial regions could affect the

467

physical state of the drug.[40] NPs may tend to aggregate, such that the drug may recrystallize and

Fig. 8. Dissolution kinetics of SD HPMCAS NPs under the accelerated aging condition in (a) FaSSGF, (b) FaSSIF and (c) FeSSIF.

ACS Paragon Plus Environment

25

ACS Applied Nano Materials 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

Page 26 of 44

468

the dissolution kinetics could be compromised.[41] Following FDA long-term stability guide,

469

harsh storage conditions are required (40 ºC, 75% RH) to demonstrate the robustness of the NP

470

formulation in those hot and humid regions. The SD HPMCAS and lecithin NPs showed a long-

471

term stability over two months under these harsh conditions by their dissolution kinetics in

Fig. 9. Dissolution kinetics of SD lecithin NPs under the accelerated aging condition in (a) FaSSGF, (b) FaSSIF and (c) FeSSIF. 472

FaSSGF, FaSSIF and FeSSIF, as shown in Figs. 8 and 9. For both HPMCAS and lecithin NPs,

473

no significant changes of release were observed between Day 1 and Month 2, showing that the

474

dissolution properties of NPs remain stable. The moisture contents of HPMCAS and lecithin

475

samples also remained constant at around 3.1 and 0.7 wt%, respectively (see SI). The sustained

ACS Paragon Plus Environment

26

Page 27 of 44 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

ACS Applied Nano Materials

476

dissolution kinetics in high temperature and humidity highlights the potential of the SD NPs for a

477

feasible commercial drug product with good long-term storage stability in developing countries.

478

Staining Test. Cryptosporidiosis is the leading cause of diarrhea in infants and small children in

479

the developing world. Therefore, it is essential to formulate an anti-Cryptosporidium therapy for

480

pediatric administration. Cfz has been widely recognized to cause exogenous pigmentation.[42] It

481

arises as a result of Cfz making contact with the oral mucosa and hence staining the mouth bright

482

red, which has a negative impact on patient compliance because of social stigma associated with

483

the red staining. In order to test the treatment compliance of our therapy, the staining effects of

484

the dried powders were quantified using polyurethane membrane to mimic human skin.[43] As

485

shown in Fig. 10, compared with Cfz powders, the SD NPs exhibit significant staining reduction

486

of 18- and 49-fold for HPMCAS and lecithin, respectively. The surface coating of HPMCAS and

487

lecithin significantly reduced mucosal adhesion, hence less staining of the membrane was

Fig. 10. (a) Photos of the stained polyurethane membrane after treatments of Cfz powder and SD HPMCAS/lecithin NPs; (b) Staining reduction with Cfz powders and SD HPMCAS/lecithin NPs. 488

observed for NPs. The SD HPMCAS powders display weaker staining reduction than lecithin

489

powders, possibly due to the carboxyl substitute cellulose, which is known to be

490

mucoadhesive.[44, 45]

ACS Paragon Plus Environment

27

ACS Applied Nano Materials 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

Page 28 of 44

491

Compressed Mini-tablet Test. As a proof of concept, we tested the suitability of the SD

492

powders to be directly compressed into mini-tablets. A mini-tablet is typically defined as a tablet

493

with a diameter less than 3 mm,[46] and desirable for pediatric and geriatric patients, providing

494

improved swallowing, flexible dosing and high degree of dispersion in the gastrointestinal tract

495

to minimize the risks of high local drug concentration. Mini-tablet production is often more

496

demanding than large tablets, and it typically requires specialized excipients to obtain the

497

targeted flow and compression properties, which may lower the drug loading and affect the

498

dissolution kinetics of the final solid powders. However, without adding extra excipients, the SD

499

powders could be compressed into mini-tablets easily. Taking SD HPMCAS NPs as an example,

500

around 7-mg powders were manufactured into one 3-mm diameter mini-tablet via simple

501

tableting procedures (See SI for the appearance of the mini-tablet). On average, 2 mL of NP

502

suspension is required and the time spent to produce one mini-tablet is approximately 1 minutes.

503

The appropriateness of flow and high compactibility of the SD powders further highlights the

504

potential of these NP powders for mini-tablet oral drug delivery. Currently, it should be noted

505

that the desired dosage form of the pediatric formulation is still under discussion, hence we have

506

not further explored the mini-tablet processing.

507

4. Conclusions

508

To demonstrate a versatile nanofabrication platform that can encapsulate drug molecules into

509

NPs and rapidly recover them from solutions without compromising the fast dissolution kinetics,

510

the FNP process was combined with spray-drying to produce dried NP powders. Taking a poorly

511

soluble, anti-Cryptosporidium drug, clofazimine, as the active ingredient, nanoparticle

512

formulations were successfully developed with two biocompatible stabilizers as surface coating,

513

HPMCAS and lecithin. NPs with both coatings demonstrated a stability window up to 6 hours

ACS Paragon Plus Environment

28

Page 29 of 44 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

ACS Applied Nano Materials

514

after formulation. Solidification of preformed NP suspensions was accomplished by

515

lyophilization and spray-drying with optimized protocols and excipients. In spray-drying, HPMC

516

E3 and mannitol were used for HPMCAS and lecithin NP formulations, respectively, to avoid

517

irreversible NP aggregations in spray-drying. The spray-dried HPMCAS NPs exhibited

518

extraordinary redispersity to nanoscale size compared with lyophilized samples, while both the

519

spray-dried and lyophilized lecithin NPs showed some aggregation during reconstitution with

520

particle sizes of O(1 µm). In addition, the spray-dried HPMCAS powders displayed a crumpled

521

morphology, but the smooth shell was formed for spray-dried lecithin powders. The further

522

characterization using XRPD and DSC demonstrated a lower order of crystallinity of Cfz in all

523

dried powders compared with raw Cfz powders, and there were no significant differences

524

observed between spray-dried and lyophilized samples for both HPMCAS and lecithin coatings.

525

The dissolution behavior of Cfz NPs was evaluated by incubation in simulated gastric conditions,

526

followed by a media swap into intestinal conditions. The newly developed Cfz formulations

527

achieved significantly higher supersaturation levels (~40-50X) in gastric fluid and faster

528

dissolution kinetics in simulated fasted or feasted fluid compared to raw Cfz powder or the

529

commercial product Lamprene. Regarding two different drying methods, the spray-dried

530

powders showed similar supersaturation and dissolution kinetics to the lyophilized samples for

531

both coatings. Moreover, even stored in high temperature and humidity, the spray-dried Cfz NPs

532

maintained complete and fast dissolutions for over two months, which highlights the excellent

533

long-term storage stability of the NP powders. The formulations also significantly reduced the

534

staining of the simulated skin samples, and were easily compressed into mini-tablets,

535

demonstrating the potential of this newly developed Cfz NPs as a feasible solid dosage form for

536

pediatric administration in global health.

ACS Paragon Plus Environment

29

ACS Applied Nano Materials 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

Page 30 of 44

537

These results highlight the potential of an integrated nanofabrication platform using FNP

538

followed by spray-drying for formulation and rapid recovery of nanoparticles with respect to

539

applications in life and materials science. In particular, a such a cost-effective process will enable

540

the easy access of nanotherapeutics for targeted patients in developing countries. However, there

541

are still questions to be further addressed. Regarding an integrated platform, an industrial-scale

542

spray drier, such as a GEA Niro Pharma Spray Drier PSD-3, can process up to 300 mL/min of

543

liquid, which would match the production rate of 160 mL/min by our FNP process. This will

544

enable a continuous process to directly produce NP powders. In addition, the difference in the

545

gut transition time and intestinal fluid composition (such as pH and bile secretion)[47] between

546

pediatric and adult populations may significantly affect the dissolution kinetics and hence the

547

bioavailability. For instance, higher gastric pH in newborns compared with adult may reduce the

548

dissolution of the weakly basic Cfz and hence the adsorption. Furthermore, understanding how

549

the dissolution kinetics relate to the bioavailability will also help find out which stabilizer and

550

processing is optimal for the desired pediatric formulations. These all requires future in vivo tests.

551 552

Acknowledgements

553

The work was supported by the Bill and Melinda Gates Foundation (BMGF, OPP1150755) and

554

the National Science Foundation Graduate Research Fellowship (DGE-1656466) awarded to

555

K.D.R. The authors thank Dr. Niya Bowers, Dr. Pius Tse, and Dr. Chih-Duen Tse for intellectual

556

discussion.

557

Supporting Information

558

Supporting Information Available: Information of AFFINSOLTM HPMCAS; Calculation of

559

effective Reynolds number for MIVM; Calculation for Pe at the spray-drying process for

ACS Paragon Plus Environment

30

Page 31 of 44 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

ACS Applied Nano Materials

560

HPMCAS and lecithin nanoparticles; Clofazimine calibration curves in THF, FaSSGF, FaSSIF

561

and FeSSIF; Residual moisture for spray-dried HPMCAS and lecithin NPs in the long-term

562

storage stability study; Appearance of lyophilized and spray-dried nanoparticles; Compressed

563

mini-tablet test.

564 565 566 567 568 569 570 571 572 573 574 575 576 577 578 579 580 581 582 583 584 585 586 587 588 589 590 591 592 593 594 595 596 597

References: 1. Savjani, K.T., A.K. Gajjar, and J.K. Savjani, Drug Solubility: Importance and Enhancement Techniques. ISRN Pharm, 2012. 2012: 195727. 2. Ku, M.S., Use of the Biopharmaceutical Classification System in Early Drug Development. Aaps J, 2008. 10: 208-212. 3. Stegemann, S., F. Leveiller, D. Franchi, H. de Jong, and H. Linden, When Poor Solubility Becomes an Issue: From Early Stage to Proof of Concept. Eur J Pharm Sci, 2007. 31: 249261. 4. Gupta, U., H.B. Agashe, A. Asthana, and N.K. Jain, Dendrimers: Novel Polymeric Nanoarchitectures for Solubility Enhancement. Biomacromolecules, 2006. 7: 649-658. 5. Abdelhamid, D., H. Arslan, Y.Y. Zhang, and K.E. Uhrich, Role of Branching of Hydrophilic Domain on Physicochemical Properties of Amphiphilic Macromolecules. Polym Chem-Uk, 2014. 5: 1457-1462. 6. Gupta, P., G. Chawla, and A.K. Bansal, Physical Stability and Solubility Advantage from Amorphous Celecoxib: The Role of Thermodynamic Quantities and Molecular Mobility. Mol Pharmaceut, 2004. 1: 406-413. 7. Matteucci, M.E., B.K. Brettmann, T.L. Rogers, E.J. Elder, R.O. Williams, and K.P. Johnston, Design of Potent Amorphous Drug Nanoparticles for Rapid Generation of Highly Supersaturated Media. Mol Pharmaceut, 2007. 4: 782-793. 8. Matteucci, M.E., J.C. Paguio, M.A. Miller, R.O. Williams, and K.P. Johnston, Flocculated Amorphous Nanoparticles for Highly Supersaturated Solutions. Pharm Res-Dord, 2008. 25: 2477-2487. 9. D'Addio, S.M., C. Kafka, M. Akbulut, P. Beattie, W. Saad, M. Herrera, M.T. Kennedy, and R.K. Prud'homme, Novel Method for Concentrating and Drying Polymeric Nanoparticles: Hydrogen Bonding Coacervate Precipitation. Mol Pharmaceut, 2010. 7: 557-564. 10. Desobry, S.A., F.M. Netto, and T.P. Labuza, Comparison of Spray-Drying, Drum-Drying and Freeze-Drying for Beta-Carotene Encapsulation and Preservation. J Food Sci, 1997. 62: 1158-1162. 11. Beck-Broichsitter, M., C. Schweiger, T. Schmehl, T. Gessler, W. Seeger, and T. Kissel, Characterization of Novel Spray-Dried Polymeric Particles for Controlled Pulmonary Drug Delivery. J Control Release, 2012. 158: 329-335. 12. Sollohub, K. and K. Cal, Spray Drying Technique: Ii. Current Applications in Pharmaceutical Technology. J Pharm Sci-Us, 2010. 99: 587-597.

ACS Paragon Plus Environment

31

ACS Applied Nano Materials 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

598 599 600 601 602 603 604 605 606 607 608 609 610 611 612 613 614 615 616 617 618 619 620 621 622 623 624 625 626 627 628 629 630 631 632 633 634 635 636 637 638 639 640

13. 14.

15.

16. 17. 18.

19.

20.

21.

22. 23. 24.

25.

26. 27.

28.

Page 32 of 44

Vehring, R., Pharmaceutical Particle Engineering Via Spray Drying. Pharm Res-Dord, 2008. 25: 999-1022. D'Addio, S.M., J.G.Y. Chan, P.C.L. Kwok, B.R. Benson, R.K. Prud'homme, and H.K. Chan, Aerosol Delivery of Nanoparticles in Uniform Mannitol Carriers Formulated by Ultrasonic Spray Freeze Drying. Pharm Res-Dord, 2013. 30: 2891-2901. Tsapis, N., D. Bennett, B. Jackson, D.A. Weitz, and D.A. Edwards, Trojan Particles: Large Porous Carriers of Nanoparticles for Drug Delivery. P Natl Acad Sci USA, 2002. 99: 12001-12005. Chaubal, M.V. and C. Popescu, Conversion of Nanosuspensions into Dry Powders by Spray Drying: A Case Study. Pharm Res-Dord, 2008. 25: 2302-2308. Cholo, M.C., H.C. Steel, P.B. Fourie, W.A. Germishuizen, and R. Anderson, Clofazimine: Current Status and Future Prospects. J Antimicrob Chemoth, 2012. 67: 290-298. Love, M.S., F.C. Beasley, R.S. Jumani, T.M. Wright, A.K. Chatterjee, C.D. Huston, P.G. Schultz, and C.W. McNamara, A High-Throughput Phenotypic Screen Identifies Clofazimine as a Potential Treatment for Cryptosporidiosis. Plos Neglect Trop D, 2017. 11. Zhang, Y.Y., J. Feng, S.A. McManus, H.D. Lu, K.D. Ristroph, E.J. Cho, E.L. Dobrijevic, H.K. Chan, and R.K. Prud'homme, Design and Solidification of Fast-Releasing Clofazimine Nanoparticles for Treatment of Cryptosporidiosis. Mol Pharmaceut, 2017. 14: 3480-3488. Johnson, B.K. and R.K. Prud'homme, Flash Nanoprecipitation of Organic Actives and Block Copolymers Using a Confined Impinging Jets Mixer. Aust J Chem, 2003. 56: 10211024. Chow, S.F., C.C. Sun, and A.H.L. Chow, Assessment of the Relative Performance of a Confined Impinging Jets Mixer and a Multi-Inlet Vortex Mixer for Curcumin Nanoparticle Production. Eur J Pharm Biopharm, 2014. 88: 462-471. Liu, Y., C.Y. Cheng, Y. Liu, R.K. Prud'homme, and R.O. Fox, Mixing in a Multi-Inlet Vortex Mixer (Mivm) for Flash Nano-Precipitation. Chem Eng Sci, 2008. 63: 2829-2842. Tanno, F., Y. Nishiyama, H. Kokubo, and S. Obara, Evaluation of Hypromellose Acetate Succinate (Hpmcas) as a Carrier in Solid Dispersions. Drug Dev Ind Pharm, 2004. 30: 9-17. Tajarobi, F., A. Larsson, H. Matic, and S. Abrahmsen-Alami, The Influence of Crystallization Inhibition of Hpmc and Hpmcas on Model Substance Dissolution and Release in Swellable Matrix Tablets. Eur J Pharm Biopharm, 2011. 78: 125-133. Zhang, K., Y.J. Xu, L. Johnson, W.Q. Yuan, Z.J. Pei, and D.H. Wang, Development of nearInfrared Spectroscopy Models for Quantitative Determination of Cellulose and Hemicellulose Contents of Big Bluestem. Renew Energ, 2017. 109: 101-109. Muller, R.H., K. Mader, and S. Gohla, Solid Lipid Nanoparticles (Sln) for Controlled Drug Delivery - a Review of the State of the Art. Eur J Pharm Biopharm, 2000. 50: 161-77. Abdelwahed, W., G. Degobert, S. Stainmesse, and H. Fessi, Freeze-Drying of Nanoparticles: Formulation, Process and Storage Considerations. Adv Drug Deliv Rev, 2006. 58: 1688-713. D'Addio, S.M., J.G.Y. Chan, P.C.L. Kwok, R.K. Prud'homme, and H.K. Chan, Constant Size, Variable Density Aerosol Particles by Ultrasonic Spray Freeze Drying. International Journal of Pharmaceutics, 2012. 427: 185-191.

ACS Paragon Plus Environment

32

Page 33 of 44 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

641 642 643 644 645 646 647 648 649 650 651 652 653 654 655 656 657 658 659 660 661 662 663 664 665 666 667 668 669 670 671 672 673 674 675 676 677 678 679 680 681 682

ACS Applied Nano Materials

29.

30.

31.

32.

33.

34. 35.

36.

37.

38.

39. 40. 41.

42. 43.

Sham, J.O.H., Y. Zhang, W.H. Finlay, W.H. Roa, and R. Lobenberg, Formulation and Characterization of Spray-Dried Powders Containing Nanoparticles for Aerosol Delivery to the Lung. Int J Pharm, 2004. 269: 457-467. Santhalakshmy, S., S.J.D. Bosco, S. Francis, and M. Sabeena, Effect of Inlet Temperature on Physicochemical Properties of Spray-Dried Jamun Fruit Juice Powder. Powder Technol, 2015. 274: 37-43. Iskandar, F., L. Gradon, and K. Okuyama, Control of the Morphology of Nanostructured Particles Prepared by the Spray Drying of a Nanoparticle Sol. J Colloid Interf Sci, 2003. 265: 296-303. Bruschi, M.L., M.L.C. Cardoso, M.B. Lucchesi, and M.P.D. Gremiao, Gelatin Microparticles Containing Propolis Obtained by Spray-Drying Technique: Preparation and Characterization. Int J Pharm, 2003. 264: 45-55. Chen, K.J., T.H. Fang, F.Y. Hung, L.W. Ji, S.J. Chang, S.J. Young, and Y.J. Hsiao, The Crystallization and Physical Properties of Al-Doped Zno Nanoparticles. Appl Surf Sci, 2008. 254: 5791-5795. Narang, A.S. and A.K. Srivastava, Evaluation of Solid Dispersions of Clofazimine. Drug Dev Ind Pharm, 2002. 28: 1001-1013. Keswani, R.K., J. Baik, L. Yeomans, C. Hitzman, A.M. Johnson, A.S. Pawate, P.J.A. Kenis, N. Rodriguez-Homedo, K.A. Stringer, and G.R. Rosania, Chemical Analysis of Drug Biocrystals: A Role for Counterion Transport Pathways in Intracellular Drug Disposition. Mol Pharmaceut, 2015. 12: 2528-2536. Kim, A.I., M.J. Akers, and S.L. Nail, The Physical State of Mannitol after Freeze-Drying: Effects of Mannitol Concentration, Freezing Rate, and a Noncrystallizing Cosolute. J Pharm Sci-Us, 1998. 87: 931-935. Clarysse, S., D. Psachoulias, J. Brouwers, J. Tack, P. Annaert, G. Duchateau, C. Reppas, and P. Augustijns, Postprandial Changes in Solubilizing Capacity of Human Intestinal Fluids for Bcs Class Ii Drugs. Pharm Res-Dord, 2009. 26: 1456-1466. Konno, H., T. Handa, D.E. Alonzo, and L.S. Taylor, Effect of Polymer Type on the Dissolution Profile of Amorphous Solid Dispersions Containing Felodipine. Eur J Pharm Biopharm, 2008. 70: 493-499. Thomas, N., R. Holm, T. Rades, and A. Mullertz, Characterising Lipid Lipolysis and Its Implication in Lipid-Based Formulation Development. Aaps J, 2012. 14: 860-871. Bott, R.F. and W.P. Oliveira, Storage Conditions for Stability Testing of Pharmaceuticals in Hot and Humid Regions. Drug Dev Ind Pharm, 2007. 33: 393-401. Kim, K.R., K.Y. Ahn, J.S. Park, K.E. Lee, H. Jeon, and J. Lee, Lyophilization and Enhanced Stability of Fluorescent Protein Nanoparticles. Biochem Bioph Res Co, 2011. 408: 225229. Muller, S., Melanin-Associated Pigmented Lesions of the Oral Mucosa: Presentation, Differential Diagnosis, and Treatment. Dermatol Ther, 2010. 23: 220-9. Tas, C., Y. Ozkan, A. Savaser, and T. Baykara, In Vitro Release Studies of Chlorpheniramine Maleate from Gels Prepared by Different Cellulose Derivatives. Farmaco, 2003. 58: 605-11.

ACS Paragon Plus Environment

33

ACS Applied Nano Materials 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

683 684 685 686 687 688 689 690 691 692 693 694 695 696

44.

45. 46.

47.

Page 34 of 44

Perioli, L., A. Ambrogi, F. Angelici, M. Ricci, S. Giovagnoli, M. Capuccella, and C. Rossi, Development of Mucoadhesive Patches for Buccal Administration of Ibuprofen. J Control Release, 2004. 99: 73-82. Zhang, Y.Y., J.W. Chan, A. Moretti, and K.E. Uhrich, Designing Polymers with Sugar-Based Advantages for Bioactive Delivery Applications. J Control Release, 2015. 219: 355-368. Lou, H., M. Liu, L. Wang, S.R. Mishra, W. Qu, J. Johnson, E. Brunson, and H. Almoazen, Development of a Mini-Tablet of Co-Grinded Prednisone-Neusilin Complex for Pediatric Use. Aaps Pharmscitech, 2013. 14: 950-958. Batchelor, H.K. and J.F. Marriott, Paediatric Pharmacokinetics: Key Considerations. Brit J Clin Pharmaco, 2015. 79: 395-404.

Table of contents

697 698 699 700 701 702 703 704 705

ACS Paragon Plus Environment

34

Page 35 of 44 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

a.

ACS Applied Nano Materials

b.

c.

Mixer

HPMCAS Lecithin (in acetone) (in tetrahydrofuran)

DI water

or

Protected nanoparticles ACS Paragon Plus Environment Clofazimine

ACS Applied Nano Materials 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

ACS Paragon Plus Environment

Page 36 of 44

Page 37 of 44

a. 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

ACS Applied Nano Materials

b.

Before SD After SD NP:E3=1:2 NP:E3=1:4 NP:E3=1:8 NP:E3=1:10

2 µm

20 µm ACS Paragon Plus Environment

ACS Applied Nano Materials

a.

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

Page 38 of 44

b.

5 um

ACS Paragon Plus Environment

10 um

50 um

50 um

Page 39 of 44 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

ACS Applied Nano Materials

ACS Paragon Plus Environment

ACS Applied Nano Materials 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

a.

b.

ACS Paragon Plus Environment

Page 40 of 44

Page 41 of 44

a.

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

ACS Applied Nano Materials

b.

ACS Paragon Plus Environment

ACS Applied Nano Materials 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

ACS Paragon Plus Environment

Page 42 of 44

Page 43 of 44

a.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Month 2 Month 1 Day 14 Day 7 Day 0

b.

ACS Applied Nano Materials

ACS Paragon Plus Environment

c.

ACS Applied Nano Materials 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

a

b

Cfz powder

SD HPMCAS NPs

SD lecithin NPs ACS Paragon Plus Environment

Page 44 of 44