Mechanisms for oral absorption enhancement of drugs by nanocrystals

Journal Pre-proof Mechanisms for oral absorption enhancement of drugs by nanocrystals Jiali Liu, Liangxing Tu, Meng Cheng, Jianfang Feng, Yi Jin

PII:

S1773-2247(19)31699-5

DOI:

https://doi.org/10.1016/j.jddst.2020.101607

Reference:

JDDST 101607

To appear in:

Journal of Drug Delivery Science and Technology

Received Date: 6 November 2019 Revised Date:

14 February 2020

Accepted Date: 17 February 2020

Please cite this article as: J. Liu, L. Tu, M. Cheng, J. Feng, Y. Jin, Mechanisms for oral absorption enhancement of drugs by nanocrystals, Journal of Drug Delivery Science and Technology (2020), doi: https://doi.org/10.1016/j.jddst.2020.101607. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier B.V.

1

Mechanisms for oral absorption enhancement of drugs by nanocrystals

2

Jiali Liu a,1, Liangxing Tu a,1, Meng Cheng a, Jianfang Feng b,a,,Yi Jin a,

3 4

a

5

Herbal Medicine, Jiangxi University of Traditional Chinese Medicine, Nanchang

6

330006, P.R. China

7

b

8

China

National Pharmaceutical Engineering Center for Solid Preparation in Chinese

School of Pharmacy, Guangxi University of Chinese Medicine, Nanning 530200, P.R.

9 10

Abstract: Currently, numerous new compounds suffer from poor water solubility,

11

hindering their oral absorption from the gastrointestinal tract and thereby limiting

12

their clinical application. Nanocrystal technology, with more than 10 products on the

13

market, is one of the favored pharmaceutical technologies for the enhancement of oral

14

bioavailability. However, this technology has a limited ability of bioavailability

15

enhancement for several drugs; therefore, a good understanding about the absorption

16

mechanisms of nanocrystals in the gastrointestinal tract is urgently needed. In this

17

review, the mechanisms of nanocrystals for improving the bioavailability of poorly

18

soluble drugs were summarized from four aspects: enhanced solubility and dissolution

19

rate, enhanced interaction with mucus layer, enhanced transport across the intestinal

20

membrane and enhanced absorption by stabilizers. In addition, the factors that impact 1

These authors contributed equally to this work. Corresponding authors at: National Pharmaceutical Engineering Center for Solid Preparation in Chinese Herbal Medicine, Jiangxi University of Traditional Chinese Medicine, Nanchang 330006, P.R. China. E-mail addresses: [email protected] (J. Feng), [email protected] (Y. Jin). 

1

21

the absorption of nanocrystals were also reviewed. We believe that this paper will

22

help scientists understand the in vivo performance of nanocrystals in the

23

gastrointestinal tract and design novel strategies for further improving the

24

bioavailability of nanocrystals.

25

Key words: Nanocrystals; Oral; Absorption; Mechanism; Transport

26 27

1 Introduction

28

Large amounts of drugs in market and newly synthesized compounds are suffering

29

the problem of poor water solubility [1-3], which attracting researches to develop

30

multiple techniques, such as salt formation [4], solubilization [5], complexation [6],

31

liposomes [7], nano-emulsion [8] and nanoparticles [9], to solve this problem. And

32

among these techniques, the nanocrystal technique has become a famous choice for

33

enhancing the bioavailability of poorly soluble drugs. Nowadays, nanocrystals have

34

exhibited great advantages on numerous drug delivery systems, such as the oral

35

delivery system [10-12], intravenous delivery system [13], transdermal delivery

36

system [14,15] and targeted delivery system [16], of which the oral administration

37

system has been the most affected. As reported, nanocrystals exhibit outstanding

38

performance on bioavailability enhancement and can increase the bioavailability of

39

poorly soluble drugs for 2- to 30-fold [17,18]. To date, there are approximately ten

40

commercial drug products based on nanocrystal technology, and more than twenty

41

products are in different clinical stages [1, 19-21]. Despite the great success in

42

business, we should bear in mind that from 2005 to the present, there have been no

2

43

products based on nanocrystals approved by the FDA for oral administration (Table 1),

44

which may be due to an insufficient enhancement of oral bioavailability. In addition,

45

many researchers have found that for some drugs, such as naproxen [22] and

46

itraconazole [23], the bioavailability only showed a less than 2-fold enhancement and

47

could hardly be further improved. Thus, a better understanding of the absorption

48

mechanisms is urgently needed.

49

The bioavailability of poorly soluble drugs is mainly determined by factors such as

50

drug absorption in the intestinal tract, drug metabolization in the liver (first-pass

51

effect), drug distribution in the blood, tissues and organs and drug excretion in bodies

52

(mainly metabolized in the liver and eliminated in the kidney). Among the influencing

53

factors mentioned above, the extent of drug absorption in the intestinal tract generally

54

plays the most important role on the oral bioavailability and drug exposure in vivo.

55

Owing to their small particle size and large surface area, nanocrystals can gain a

56

higher dissolution rate and water solubility compared to coarse drugs, thus leading to

57

an enhancement of bioavailability. However, in recent years, some other absorption

58

mechanisms have been found, and the absorption mechanisms of nanocrystals have

59

not yet been completely reviewed.

60

In this paper, we will systematically review the absorption mechanisms of

61

nanocrystals from general aspects, such as enhanced solubility and dissolution rate, to

62

less studied aspects, such as enhanced mucoadhesion, enhanced diffusion in mucus

63

layer, and finally, to the knowledge gained in the last decade about transport

64

mechanisms across the epithelial membrane. Meanwhile, the factors that influence the

3

65

absorption of nanocrystals will also be summarized and reviewed. We believe that this

66

review will help scientists understand the in vivo performance of nanocrystals in the

67

gastrointestinal tract and design novel strategies for further improving the

68

bioavailability of nanocrystals.

69 70

2 Oral absorption mechanisms in gastrointestinal tract

71

After oral administration, nanocrystals enhance the bioavailability of poorly

72

water-soluble drugs by undergoing complex absorption mechanisms for the transport

73

of drugs or intact nanocrystals from the gastrointestinal tract to the blood or lymphatic

74

system (Table 2). The absorption mechanisms mainly include enhanced solubility and

75

dissolution rate, enhanced interaction with mucus layer, enhanced transport across the

76

intestinal membrane and enhanced absorption by stabilizers.

77

2.1 Enhanced saturation solubility and dissolution rate

78

After oral administration, the drug should dissolute from the formulation into the

79

gastrointestinal juice, and then transport across the gastrointestinal epithelium. Owing

80

to the particle size in nanoscale, nanocrystals are considered to enhance the saturation

81

solubility and dissolution rate of drugs, hence forming a higher drug concentration in

82

the unstirred water layer (mucosa), and excluding the diffusion ability of nanocrystals

83

(or dissolved drug) in mucosa, higher drug concentration gradient between the

84

gastrointestinal membrane and blood vessels could gained and the bioavailability of

85

drugs could enhanced thereafter.

86

The increased saturation solubility can be explained by Ostwald–Freundlich’s

4

87

equation (Equation 1) [36], and according to the equation, a decreased particle size

88

leads to increased saturation solubility.

89

log

CS C∞

=

2σV

(Equation 1)

2.303RTρr

90

where Cs is the saturation solubility, C∞ is the solubility of the drug consisting of

91

large particles, σ is the interfacial tension substance, V is the molar volume, R is the

92

gas constant, T is the absolute temperature, ρ is the density of the drug and r is the

93

radius of the drug particle.

94

An increased dissolution rate as the particle size is reduced can also be observed

95

after transferring drugs into nanocrystals, and it can be explained by the

96

Noyes-Whitney equation (Equation 2).

97

dC dt

=

DS h

(Cs −

Xd V

)

(Equation 2)

98

where dC/dt is the dissolution rate, D is the diffusion coefficient, S is the surface

99

area, h is the diffusional distance, Cs is the saturation solubility and Xd/V is the

100

concentration around the particles.

101

According to this equation, the dissolution rate will be enhanced when the

102

diffusional distance is decreased, which has a positive correlation with particle size

103

(as shown in the Prandtl boundary layer equation) (Equation 3) [37,38].

104

hH = k(L1⁄2 /V 1⁄2 )

(Equation 3)

105

where hH is the hydrodynamic boundary layer, k denotes a constant, L is the length

106

of the particle surface and V is the relative velocity of the flowing liquid surrounding

107

the particle.

108

Therefore, owing to their small particle size and large surface area, nanocrystals

5

109

can gain a higher dissolution rate and water solubility than coarse drugs, thus leading

110

to an enhancement in the bioavailability of poorly soluble drugs.

111 112

2.2 Enhanced interaction with mucus layer

113

2.2.1 Enhanced mucoadhesion to gastrointestinal mucosa

114

Mucosa is the moist surface lining the walls of the gastrointestinal tract, and its

115

moistness is usually caused by the presence of a mucus layer. The major functions of

116

mucosa are protection and lubrication [39]; however, these functions can block the

117

contact of drugs and epithelial cells, thus hindering the absorption of drugs. The

118

formation of mucoadhesion can be explained by six general theories: 1) the electronic

119

theory (electrostatic attraction forces between the drug and mucus) [40], 2) the

120

wetting theory (the spontaneous spreading of mucus onto the particle surface) [41], 3)

121

the adsorption theory (the attachment of adhesives on the basis of hydrogen bonding

122

and van der Waals’ forces) [39], 4) the diffusion theory (interpenetration of polymer

123

chains and mucus) [42], 5) the mechanical theory (interlocking or interaction between

124

mucus and the irregular or enlarged surface of the particle) [43], and 6) the fracture

125

theory (strength of adhesive forces required for the detachment of the drug and mucus)

126

[44].

127

Due to their small particle size and potential use of functional stabilizers (especially

128

for stabilizers with positive charges, which could increase the electrostatic

129

interactions between nanocrystals and mucosa, as mucosa exhibit negative charge

130

profile) [45], nanocrystals can exhibit higher electrostatic attraction forces, van der

6

131

Waals’ forces and surface area (highly associated with spontaneous spreading,

132

interpenetration and interlocking or interaction between mucus and particles), which

133

can lead to enhanced mucoadhesion to mucosa [46] and the prolonged retention time

134

of drugs, hence improving the absorption and bioavailability of poorly soluble drugs.

135

However, what should be noticed is that too strong mucoadhesion can interrupt the

136

diffusion of nanocrystals in mucus layer, thus hindering the contact between

137

nanocrystals and epithelial membrane, and leading to decrease in drug absorption

138

[47].

139

2.2.2 Enhanced diffusion in mucus layer

140

When contacting with mucosa, drugs are surrounded by mucus layer, a dynamic,

141

semipermeable barrier with thickness about 120-830 µm in rat’s intestinal tract [48],

142

that exist in a variety of organs or tissues like gastrointestinal tract and nasal cavity

143

[49]. The mucus layer is composed of mucin glycoproteins, lipids, inorganic salt and

144

water, and could enable the exchange of nutrients, water and gases and is

145

impermeable to bacteria and pathogens. As foreign functional substances, the drugs

146

may be identified as “harmful” materials by mucus and trapped or immobilized before

147

they contact the epithelial surface [50].

148

The main structural component of

the mucus layer is mucin, a highly

149

glycosylated protein, which can physically and chemically interact with each other or

150

with other components in mucus layer to form a mesh-like structure (average pore

151

size 10-500 nm), and this mesh-like structure can regulate the drug and particle

152

diffusion to the underlying epithelium [51]. Compared to bulk drugs, nanocrystals can

7

153

generate a higher penetration ability across the mucus layer by forming a reservoir to

154

release the drugs with a smaller effective diffusion thickness and by using

155

surface-altering agents (as a stabilizer), thus avoiding mucus adhesion and rapid

156

mucus clearance and improving the contact between the drugs and the IECs (Fig.1).

157

One classic example is lovastatin (LOV): after being transformed into rod shaped

158

nanocrystals (RNCs), the Papp value in mucus improved from 4.39  10-6 cm/s for the

159

LOV solution to 6.21  10-6 cm/s for RNCs, indicating that the nanocrystals more

160

easily penetrated the mucus layer [52]. In another study, Ueda and his colleagues

161

constructed fenofibrate nanocrystals by employing Hypromellose (HPMC), a material

162

that functions on mucin diffusion, as a stabilizer. Mucin is a mixture of glycosylated

163

proteins and is the main component of the mucus layer, so the nanocrystals improved

164

the permeability of fenofibrate through the mucin layer. In addition, the authors also

165

found that HPMC with a lower molecular weight enhanced the flexibility of the

166

nanocrystal interface and inhibited its interaction with mucin, leading to a faster

167

diffusion of nanocrystals through mucin [53].

168 169

2.3 Enhanced transport across the intestinal membrane

170

Nanocrystals are generally assumed to dissolve more than coarse drugs, thus

171

forming a higher drug concentration gradient, and leading to improved absorption

172

from the gastrointestinal tract. With this knowledge, the volume of water in the

173

intestinal tract, excluding the effects of pH, bile salts and emptying time, influences

174

the dissolution rate and the oral absorption of drugs. However, the water content in

8

175

the

intestinal

tract

is

highly

variable,

which

depending

on

the

176

physiological/pathological conditions and/or fasted/fed state [54]. It has been reported

177

that in human volunteers, the fasted stomach contains approximately 35 mL of resting

178

water, and the intestinal tract contains approximately 77 mL of water distributed into

179

16 pockets of ~5 mL each. Meanwhile, after drinking 240 mL water, the gastric water

180

volume declined rapidly with a half emptying time of 13 min [55]. Considering that

181

the water in the gastrointestinal tract may be insufficient for dissolving nanocrystals

182

instantaneously upon oral administration, there may exist absorption mechanisms

183

related to the interaction between nanocrystals and the epithelial membrane. To date,

184

studies on other absorption mechanisms that happen on the epithelial membrane are

185

limited, but several studies have exhibited new views toward understanding the

186

performance of nanocrystals in the intestinal tract. In these studies, mechanisms such

187

as endocytosis pathways and M cell uptake were observed (Fig.2), and with the help

188

of these mechanisms, the nanocrystals could further improve the absorption of poorly

189

soluble drugs.

190

2.3.1 Endocytosis pathways

191

The question that whether nanocrystals can transport across epithelial cells intact or

192

not has attracted scientists for many years, and researchers have proved that

193

endocytosis is the major enter-cellular mechanism. To estimate the potential existence

194

of endocytosis on the transport of nanoparticles, Müller, et al. proposed the

195

conception of the nanotoxicological classification system, and according this system,

196

particles larger than 100 nm cannot be taken up by cells, while particles smaller than

9

197

100 nm can be internalized into cells via endocytosis [56]. However, this predictive

198

system does not seem applicable for nanocrystals, as some nanocrystals larger than

199

100 nm, e.g., curcumin nanocrystals with particle sizes of 321 nm [57] and

200

nimodipine nanocrystals with diameters of 833.3 nm [58], have been taken up by

201

enterocytes via different mediated routes of endocytosis. The endocytosis pathways

202

currently found were caveolin-mediated [58,59], caveolae-mediated [59], lipid

203

draft-mediated [60] and macrophage-mediated endocytosis [58,61-63], and cav-1,

204

dynamin and actin filaments modulated the endocytosis process [64].

205

Nanocrystals in cells cannot enhance the absorption of drugs until they transport

206

through the inner medium and release outside of the cells. Exocytosis, transcytosis

207

and intracellular trafficking happen after the nanocrystals enter the cells. Some

208

researchers have found that besides being present in the endocytosis process, lipid

209

draft also exists in the transcytosis and exocytosis of nanocrystals [60]. Several other

210

studies have revealed that Golgi complexes, lysosomes and endosomes participate in

211

the processes of transcytosis and intracellular trafficking [45,60,64].

212

2.3.2 M cell uptake pathways

213

Microfold (M) cells are locating in the follicle-associated epithelium (FAE) of

214

Peyer’s patches and in the gut-associated lymphoid tissue (GALT), which are

215

components of the mucosal immune system. Compared to epithelial cells, M cells

216

have less glycocalyx and reduced protease activity, which can benefit the transport of

217

nanocrystals through the intestinal membrane. Despite the amount of M cells in the

218

human gastrointestinal tract is low (5% of human FAE and less than 1% of the total

10

219

intestinal surface) [65,66], M cells have a strong transport capacity for many types of

220

materials, such as bacteria, viruses and antigens. As for nanocrystals or nanoparticles,

221

the absorption proportion through M cell-mediated pathways is still unclear. As

222

estimated by Yu, et al. [67], the transport of nanoparticles or nanocrystals through M

223

cells can be extremely low, as a result of the lack of specificity of nanoparticles

224

toward M cells and the capture of nanoparticles by macrophages and dendritic cells

225

(which limits the efficacy of nanoparticle entry into the bloodstream). Recently,

226

several studies have reported that M cells may play a relatively important role on the

227

transport of nanocrystals. According to Liu and colleagues [68], intact carvedilol

228

nanocrystals may be directly taken up by M cells, and the absorption of carvedilol

229

will be improved thereafter. Fu et al. [58] found that nimodipine nanocrystals were

230

taken up by M cells and then drained into the mesenteric lymph duct, thus avoiding

231

the first-pass metabolism and resulting in enhanced bioavailability. In Shen’s study

232

[69], it was reported that M cells recognized and transported foreign particulates from

233

the lumen to basolateral lymphoid tissues, and the M cell-mediated route was

234

involved in the absorption of integral quercetin hybrid nanocrystals.

235 236

2.4 Other mechanisms mainly contributed to stabilizers in nanocrystals system

237

Stabilizer is an important part in nanocrystals system, and excluding the absorption

238

enhancing mechanisms of nanocrystals showed above, there are several other

239

mechanisms that mainly contributed to stabilizers, and these mechanisms are surely

240

important for the absorption enhancements of drugs via nanocrystals technology.

11

241

2.4.1 Inhibited p-gp efflux

242

P-glycoprotein (P-gp) widely exists on the surface of IECs, and its major function

243

is to protect the body by identifying and pumping out foreign substances that have

244

been transported across the cell layer. However, the efflux effect can interrupt the

245

absorption of drugs if they are P-gp substrate. It seems impossible that the production

246

process of nanocrystals has the ability of changing a P-gp substrate-like drug into an

247

insensitive one, but when constructed with P-gp efflux inhibitors, the fate of

248

nanocrystals will change in the presence of the P-gp efflux inhibitors (Fig.3). Sharma,

249

et al. [70] found that Tween 80 has a P-gp inhibition effect, and paclitaxel

250

nanocrystals prepared with Tween 80 improved the bioavailability of paclitaxel

251

approximately 12.5-fold compared to that of paclitaxel crystals. To further improve

252

the bioavailability, this group synthesized Pluronic-g-Cationic polyelectrolyte as a

253

functional stabilizer to form novel nanocrystals. By using the effects of P-gp efflux

254

inhibition and opening tight junctions of this stabilizer, the bioavailability of the

255

nanocrystals was 12.6-fold that of Taxol™, a commercial product of paclitaxel [71].

256

2.4.2 Paracellular pathways

257

Paracellular pathways are normally restricted by tight junctions, which are locating

258

at the outermost end of the intercellular space and mediate the transport of substances

259

across the epithelium by passage through the intercellular space between intestinal

260

epithelial cells. In general, tight junctions allow the passage of small substrates, such

261

as ions and electrolytes [72-74], but prevent the transport of large molecules with a

262

molecular radius exceeding 15 A°, because the dimension of the paracellular space in

12

263

the natural state ranges from 0.3 to 1.0 nm [75]. The paracellular space will increase

264

to 20 nm when it is fully opened; however, this space is still too narrow for most

265

nanocrystals to penetrate. Therefore, it is considered that the transport of nanocrystals

266

across the epithelial membrane via the paracellular pathway is severely restricted

267

[67,76], and indeed, several studies have reported that the paracellular pathway does

268

not exist in the transport process of nanocrystals [55,58,64]. However, we should bear

269

in mind that nanocrystals with a measured particle size (mean diameter or D50) below

270

100 nm or even 50 nm will have a significant quantity of particles with a diameter

271

below 20 nm or even smaller [77], and the paracellular pathway may exist in the

272

transport of ultra-small nanocrystals in cases where the tight junctions were opened by

273

functional materials.

274

To date, no work has reported that nanocrystals can open TJs without the use of

275

functional stabilizers. However, several researchers have proven that TJs can be

276

opened with the use of functional materials; for example, Quan, et al. [78] modified

277

the surface of nitrendipine nanocrystals with chitosan, a cationic polymer with a

278

positive charge, and they found that the modified nanocrystals could improve the

279

bioavailability approximately 1.4-fold compared with the initial nanocrystals, as

280

chitosan could regulate the TJs by inducing changes in transmembrane CLDN4

281

protein [79].

282 283 284

13

285

3 Factors influencing the absorption of nanocrystals

286

3.1 Particle size

287

Particle size undoubtedly plays an important role in the absorption of nanocrystals,

288

and the function of particle size on the transport of nanocrystals can be summarized as

289

follows: a) influences the saturation solubility and dissolution rate; b) influences the

290

mucoadhesion of nanocrystals (smaller particle sizes create an enhanced contact area

291

with gastrointestinal mucosa, thus enhancing the mucoadhesion); c) influences the

292

diffusion in mucus layer); d) influences the endocytosis pathway (small particle sizes

293

seem to benefit endocytosis); e) influences the paracellular pathway (ultra-small

294

(below 100 or 50 nm) nanocrystals may be partly transported by the paracellular

295

pathway).

296

Simply put, it is considered that the smaller the particle size, the greater the

297

absorption. To date, many researchers have proven the increase of bioavailability with

298

the decrease of particle sizes of nanocrystals (Fig.4). Xia et al. found that the

299

bioavailability of nitrendipine increased as the particle sizes of nanocrystals decreased

300

[80]. Another study observed a 4.4-, 4.7-, 5.1- and 7.3-fold increase in bioavailability

301

with nanocrystals with particle sizes of 700 nm, 400 nm, 120 nm and 80 nm,

302

respectively, when compared to that of coarse coenzyme Q10 [81]. The bioavailability

303

of nisoldipine was found to be enhanced 2.2-, 5.1- and 7.1-fold after the particle sizes

304

of nisoldipine decreased from 7.3 µm to 1.2 µm, 473 nm and 240 nm, respectively

305

[29]. Regarding puerarin, a poorly water-soluble compound extracted from Pueraria

306

thunbergiana Benth, the bioavailability was increased 1.5-fold (1.9 µm) and 7.6-fold

14

307

(526 nm) compared to crude puerarin (≈20 µm) [82]. After decreasing the particle size

308

of nimodipine, the bioavailability of nimodipine nanocrystals was approximately

309

1.5-fold and 3.7-fold that of nimodipine (16.3 µm) for nanocrystals with particle sizes

310

of 4.1 µm and 833 nm, respectively [83].

311 312

3.2 Zeta potential

313

Zeta potential represents the strength of the charge on the surface of particles, and a

314

high absolute value (generally above 30 mV) [84] is generally required for

315

stabilization of the nanocrystal system, in which electrostatic repulsion is the only

316

stable mechanism. In addition to the influence on the stability of nanocrystals, zeta

317

potential has a mucoadhesive effect on the absorption of nanocrystals.

318

It is well known that the process of mucoadhesion is a consequence of interactions

319

between the mucus layer on mucosa and mucoadhesive particles, so this process is

320

greatly dependent on the mucoadhesive strength between mucosa and particles. As the

321

charge on the particle surface generates electrostatic forces and zeta potential

322

represents the strength of the charge, zeta potential has a fine positive correlation with

323

mucoadhesive strength [85]. Despite the rarity of studies on the correction of zeta

324

potential with mucoadhesion or the bioavailability of nanocrystals, we can still

325

speculate that a higher absolute zeta potential benefits the mucoadhesion of

326

nanocrystals to the gastrointestinal tract, hence favoring the enhancement of oral

327

absorption. In addition, considering that the gastrointestinal mucus layers are

328

negatively charged at a neutral pH [86], nanocrystals with positive zeta potential

15

329

could be more favor the attachment of nanocrystals and mucosa than that’s with

330

negative zeta potential [87,88].

331 332

3.3 Crystalline state

333

The crystalline state, which is caused by a phenomenon of polymorphs of materials,

334

is an important part of crystal studies. In general, crystalline polymorphs have the

335

same chemical composition but different crystal structures, which may cause

336

differences in lattice structures and/or molecular conformations; therefore, they

337

possess different physicochemical and thermodynamic properties, such as energy,

338

melting point and solubility [89,90]. As a typical feature of crystals, drug nanocrystals

339

possess a definite crystalline state (may be amorphous, crystal form A, crystal form B

340

crystal form C, etc.), which may differ depending on the type of crystalline state of

341

the initial drug. As has been reported, many drugs exhibit polymorphs, thus leading to

342

different nanocrystals with different crystalline states. The differences in crystalline

343

states may cause variations in bioavailability. The effect of polymorphs on the

344

bioavailability of poorly soluble drugs has already been reviewed [91], and several

345

drugs, such as carbamazepine [92,93] and phenylbutazone [94], have exhibited

346

differences in bioavailability between different crystalline states. In addition,

347

nanocrystals may present as completely crystalline, partially crystalline or completely

348

amorphous, and amorphous nanocrystals, with their high saturation solubility, are

349

more ideal for improving the bioavailability of poorly water-soluble drugs. However,

350

owing to their high surface energy, amorphous or partially amorphous nanocrystals

16

351

often bear the risk of re-crystallization, which could lead to a decrease in

352

bioavailability [95].

353 354

3.4 Shape of nanocrystals

355

As a typical type of crystal, nanocrystals can form numerous shapes, such as

356

slice-like [96], oval-like [97], rod-like [98], sphere-like [99], needle-like [27],

357

granule-like [100] and irregularly shaped [101], under different crystallization

358

conditions. According to the theory of Heywood’s shape factor [102], the shape

359

impacts the surface area by changing the surface to volume shape factor, which is

360

shown in the equation below (Equation 4):

361

𝑆𝑣 =

𝛼𝑠𝑣

(Equation 4)

𝑑𝑠𝑣

362

where Sv represents the volume-specific surface area, dsv is the volume-specific

363

mean diameter and αsv denotes the surface to volume shape factor. The shape factor

364

value alters as the shape of the particle transforms, and the minimum value for the

365

shape factor is 6, which refers to spherical particles, so the shape factor value and

366

surface area increase for particles that deviate from the spherical shape. In addition,

367

the shape factor also has a negative correlation with the diffusion layer thickness,

368

meaning that the diffusion layer thickness decreases as the shape factor value

369

increases [103].

370

Differences in shapes may cause differences regarding the in vivo performance of

371

nanocrystals, and the influence of different shapes on the absorption of nanocrystals

372

has been studied by several groups. For instance, Guo et al. [103], constructed rod

17

373

shaped and spherical-like lovastatin nanocrystals with similar diameters, and found

374

that rod-like crystals had a larger surface area and smaller diffusion layer thickness

375

than spherical crystals, leading to a faster dissolution rate and higher Cmax and

376

bioavailability (rod-like nanocrystals showed a 1.5-fold increase in Cmax and a

377

1.36-fold increase in AUC0-24

378

studies [52], the effects of particle shapes on mucus permeation, transepithelial

379

transport and bioavailability were investigated by using spherical, rod shaped and

380

flaky lovastatin nanocrystals (SNCs, RNCs, and FNCs, respectively). The results

381

showed that the RNCs exhibited the best ability for mucus permeation, cellular uptake

382

and the transmembrane transport of nanocrystals, and the AUC0-24h of RNCs was

383

1.44-fold and 1.8-fold higher than that of SNCs and FNCs, respectively.

h

compared to spherical nanocrystals). In follow-up

384 385

3.5 Stabilizers

386

To best apply their potential for enhancing bioavailability, nanocrystals should be

387

stabilized in gastrointestinal tract juice, and the major function of stabilizers is

388

certainly to stabilize the nanocrystal system. The major stabilization mechanisms of

389

stabilizers can be summarized as the electrostatic repulsion effect and the steric

390

stabilization effect [104]. When using ionic stabilizers, such as sodium dodecyl sulfate

391

and chitosan, the electrons from the stabilizers can be adsorbed onto the surface of

392

nanocrystals and cause them to have a certain zeta potential, thus stabilizing the

393

system via the electrostatic repulsion effect. When using non-ionic stabilizers, such as

394

Hypromellose and povidone, the stabilizers adsorb onto the surface of nanocrystals

18

395

and then form a steric layer, subsequently hindering the contact between nanocrystals,

396

and hence stabilizing the system via the steric stabilization effect (Fig.5).

397

Meanwhile, stabilizers can play other roles in the absorption of nanocrystals. Many

398

researches have revealed that stabilizers affect the absorption process of nanocrystals

399

or other drug delivery systems (Table 3) and may function through mechanisms such

400

as: a) increasing the saturation solubility and dissolution rate, b) enhancing

401

mucoadhesion, c) opening tight junctions and enhancing permeability, d) inhibiting

402

the P-gp efflux, and e) enhancing cellular uptake.

403

Considering the statements mentioned above, we can realize that the absorption of

404

nanocrystals is influenced by multiple factors through different mechanisms, and the

405

relationships between the influencing factors and the absorption mechanisms are

406

shown in Fig. 6.

407 408

4 Conclusion

409

Compared to other nanocarrier drug delivery systems, nanocrystals are an

410

important option for enhancing the bioavailability of poorly water-soluble drugs, and

411

all drugs can be transformed into nanocrystals; therefore, this technology can be

412

applied to all poorly soluble drugs. In the last few decades, nanocrystals have gained

413

great attention and exhibited huge advantages, such as their ease to produce and high

414

drug loading. There are currently more than ten products on the production pipeline,

415

and majority of them are for oral administration. However, nanocrystals encounter a

416

big challenge with enhancing bioavailability on a large scale (e.g., above 4-fold), and

19

417

despite absorption mechanisms being partially reviewed by some papers, the

418

comprehensive insight about nanocrystals in the intestinal tract is still lacking. To sum

419

up the research on nanocrystals, scientists have payed increasing attention to the

420

absorption mechanisms of nanocrystals, and the mechanisms they have studied range

421

from general aspects, such as enhanced solubility and dissolution rate, to cellular level

422

mechanisms, such as endocytosis and M cell uptake pathways. The absorption of

423

nanocrystals from the gastrointestinal tract to blood vessels is a complex process with

424

multiple mechanisms at work, and studies on the absorption mechanisms, especially

425

mechanisms at the cellular level or even the molecular level, are rare, so researchers

426

should pay more attention to them in the future. In this review, we comprehensively

427

summarized the absorption mechanisms of nanocrystals that have been identified in

428

the last decade, and we believe a better understanding of the in vivo performance of

429

nanocrystals in the gastrointestinal tract benefits the design of novel strategies for

430

further improving the bioavailability of nanocrystals.

431 432

Acknowledgements

433

This work was supported by the National Natural Science Foundation of China

434

(81960717, 81573623), the Natural Science Foundation of Jiangxi Province

435

(20192BAB215057), the “Double First-Class” Discipline Project of Jiangxi Province

436

(JXSYLXK-ZHYA0015) and the PhD startup foundation of Jiangxi University of

437

TCM (2018BSZR018).

438

20

439

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Fig.1. Faster diffusion through mucus layer of nanocrystals lead to enhancement of absorption in gastrointestinal membrane compared to crude drug (microparticle).

Fig.2. The cellular transport mechanisms of nanocrystals across the intestinal membrane. Nanocrystals could be taken up by epithelial cells via caveolin-mediated, caveolae-mediated, lipid draft-mediated and macrophage-mediated endocytosis, and cav-1, dynamin and actin filaments modulated the endocytosis process. The entered nanocrystals could transport across the epithelial cell with the aid of lipid draft, Golgi complexes, lysosomes and endosomes. In addition, nanocrystals could be taken up by M cells in Peyer’s patches, and be transported to lymph-vessel.

Fig.3. The inhibition effect of P-gp efflux in the absorption process of nanocrystals. The nanocrystals itself has no inhibition effect of P-gp efflux, however, with the help of functional stabilizers (inhibitor of P-gp efflux), the nanocrystals could inhibit the P-gp efflux, hence improving the transport of drugs across epithelial membrane.

Fig.4. The effect of particle sizes on the bioavailability of nanocrystals.

Fig.5. The stable mechanisms of stabilizers in nanocrystals system.

Fig.6. The relationships between influence factors and absorption mechanisms.

Table 1 Typical products based on nanocrystals approved by FDA. Approval year

Wet milling

Administration route Oral

Anti-psychotic

Wet milling

Oral

2001

Morphine sulfate

Anti-chronic pain

Wet milling

Oral

2002

Ritalin LA®/Novartis

Methylphenidate HCl

Anti-psychotic

Wet milling

Oral

2002

Zanaflex CapsulesTM/Acorda

Tizanidine HCl

Muscle relaxant

Wet milling

Oral

2002

Emend®/Merck

Aprepitant

Antiemetic

Wet milling

Oral

2003

Tricor®/Abbott

Fenofibrate

Hypercholesterolemia

Wet milling

Oral

2004

Megace® ES/Par Pharma

Megestrol acetate

Appetite stimulant

Wet milling

Oral

2005

Triglide™/Skye Pharma

Fenofibrate

Hypercholesterolemia

HPH

Oral

2005

Invega Sustenna®/Johnson

Tizanidine HCl

Anti-depression

HPH

Injection (i.m.)

2009

Invega Trinza® / Johnson

Tizanidine HCl

Anti-depression

HPH

Injection (i.m.)

2015

Aristada® /Alkermes

Tizanidine HCl

Schizoprenia

HPH

Injection (i.m.)

2015

Product/Company

Drug Compound

Clinical using

Production approach

Rapamune®/Wyeth

Sirolimus

Immunosuppressive

Focalin XR®/Novartis

Dexmethylphenidate HCl

Avinza®/King Pharma

*HPH: high pressure homogenization

2000

Table 2 Changes of pharmacokinetic parameters by several absorption mechanisms of nanocrystals Drugs

Icaritin

Aceclofenac

Aceclofenac

Prepared methods Precipitation– ultrasonication

Precipitation– ultrasonication

Wet milling

Dosage form (particle size)

Control (particle size)

Mechanisms

NCs (220 nm)

Coarse suspension

NCs (112 nm)

Coarse suspension and marketed tablets

NCs (485 nm)

Coarse suspension

NCs (460 nm)

Coarse suspension (80 µm)

Enhanced dissolution rate Enhanced dissolution rate

Fenofibrate

Probe sonication

Puerarin

High pressure homogenization

SNCs (229 nm)

Coarse capsule (20 µm)

Nisoldipine

Media milling

NCs (1227-240 nm)

Coarse suspension (7.3

Enhanced dissolution rate 2.6-4.5-fold increase in solubility, enhanced dissolution rate ≈2.0-fold increase in solubility, 2.1-fold increase in dissolution rate 4.5-11.2-fold increase in solubility, enhanced dissolution rate

Changes of pharmacokinetic parameters

Animals

References

SD Rats

[24]

Swiss albino rabbits

[25]

1.9-fold increase in Cmax, 1.6-fold increase in bioavailability.

Wistar Rats

[26]

4.7-fold increase in Cmax, 4.7-fold increase in bioavailability.

New Zealand white rabbits

[27]

Beagle dogs

[28]

SD Rats

[29]

4.7-fold increase in Cmax, 2.0-fold increase in bioavailability, 5.9-fold decrease in Tmax 2.8-5.0-fold increase in Cmax, 2.1-3.9-fold increase in bioavailability, 2.0-fold decrease in Tmax

3.2-fold increase in Cmax, 4.5-fold increase in bioavailability. 4.4-25.1-fold increase in Cmax, 3.1-6.2-fold increase

µm)

Resveratrol

Precipitation

NCs (222 nm)

Coarse suspension

Enhanced dissolution rate

Breviscapine

High pressure homogenization

NCs (≈140 nm)

Coarse suspension (30.62 µm)

Enhanced dissolution rate

20(S)-Protopanaxadiol

Precipitation

NCs (90 nm)

Coarse suspension

Enhanced solubility and dissolution rate

Coarse suspension and NCs without chitosan

Enhanced mucoadhesion and permeability

Diacerein

Sonoprecipitation

NCs (≈150 nm)

Carvedilol

Precipitation– ultrasonication

Buccal films with NCs (495 nm)

Marketed tablets

Enhanced mucoadhesion and permeability

Ursolic acid

High pressure homogenization

NCs (291 nm) and microcrystals (1299 nm)

Coarse suspension

Enhanced mucoadhesion and dissolution rate

NCs: nanocrystals; SNCs: solid nanocrystals

in bioavailability. 2.0-fold decrease in Tmax 3.1-fold increase in Cmax, 3.5-fold increase in bioavailability. 4.1-fold increase in Cmax, 4.5-fold increase in bioavailability. 4.7-fold increase in Cmax, 1.5-fold increase in bioavailability. 1.4-0.5-fold increase in Cmax, 1.7-1.2-fold increase in bioavailability, 2.1-12.0-fold decrease in Tmax. 29.3-fold increase in Cmax, 9.2-fold increase in bioavailability, 2.0-fold decrease in Tmax. 2.6-1.4-fold increase in bioavailability.

SD Rats

[30]

Wister Rats

[31]

SD Rats

[32]

Wister Rats

[33]

Rabbits

[34]

SD Rats

[35]

Table 3 The effects of stabilizers on the in vivo performance of nanocrystals. Stabilizers

Mechanisms

Drugs

Enhanced in vivo performance

References

Enhanced mucoadhesion

Diacerein

Delayed Tmax and 1.22-fold increase in bioavailability to control

[33]

Opening TJs

Endotoxins,

Enhanced absorption

[105]

Opening TJs

Insulin

1.8-fold increase in bioavailability to control

[106]

Enhanced mucoadhesion and opening TJs

Nitrendipine

Delayed Tmax and 1.4-fold increase in bioavailability to control

[78]

Enhanced cellular uptake

paclitaxel

Enhanced bioavailability and therapeutic effect

[107]

Pluronic-grafted chitosan

P-gp inhibition and opening TJs

paclitaxel

12.6-fold increase in bioavailability to TaxolTM

[71]

Trimethyl chitosan

Enhanced cellular uptake

gemcitabine

5.5-fold increase in bioavailability to coarse drug

[108]

P-gp inhibition

Ezetimibe

Enhanced therapeutic effect

[109]

P-gp inhitition

Berberine chloride

1.9-fold increase in bioavailability to control

[110]

P-gp inhibition and enhanced solubility and dissolution rate

Andrographolide

1.23-fold increase in Cmax and 1.7-fold increase in bioavailability to control

[111]

Enhanced solubility

Paclitaxel

6.3-fold increase in bioavailability to coarse drug

[112]

Enhanced solubility and dissolution rate

Cyclosporin A

1.5-fold increase in Cmax and 1.7-fold increase in bioavailability to coarse drug

[113]

Chitosan

D-α-tocopheryl polyethylene glycol 1000 succinate (TPGS) Sodium dodecyl sulfate (SDS)

Enhanced permeability/transport

Fenofibrate

1.2-fold increase in bioavailability to control

[114]

Enhanced permeability/transport

Danazol, fenofibrate and itraconazole

Enhanced bioavailability to coarse drug

[115]

Eudragit RLPO

Enhanced mucoadhesion

Glimepiride

2.04-fold increase in bioavailability to coarse drug

[86]

Tween 80

P-gp inhibition

Digoxin

1.61-fold increase in bioavailability to control

[116]

Poloxamer

Enhanced cellular uptake

Carvedilol

1.9-4.9-fold increase in bioavailability to coarse drug

[117]

Sodium poly styrene sulfonate

Enhanced mucoadhesion

Paclitaxel

14.9-fold increase in bioavailability to coarse drug

[70]

HM30181

P-gp inhibition

Paclitaxel

6.3-fold increase in bioavailability to control and Fa=25.8%

[118]

Soluplus®

Control: the same formulation without functional stabilizer; TJs: tight junctions

In this paper, the mechanisms of nanocrystals in improving bioavailability of poor soluble drugs were summarized from six aspects: enhanced solubility and dissolution rate, enhanced mucoadhesion, enhanced permeability, inhibited P-gp efflux, decrease fasted/fed variation, and the transport mechanisms across epithelial membrane. In addition, the factors impacted the absorption of nanocrystals were also reviewed.

Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: