Developing nanocrystals for cancer treatment...Drug nanocrystals, on the other hand, may be injected intravenously without resorting to any car-rier chemicals for encapsulation and/or

Nanomedicine (Lond.) (Epub ahead of print) ISSN 1743-5889

part of

Review

10.2217/NNM.15.73 © 2015 Future Medicine Ltd

Nanomedicine (Lond.)

Review 2015/07/3010

15

2015

Nanocrystals are carrier-free solid drug particles that are sized in the nanometer range and have crystalline characteristics. Due to high drug loading (as high as 100%) – free of organic solvents or solubilizing chemicals – nanocrystals have become attractive in the field of drug delivery for cancer treatment. Top–down and bottom–up approaches have been developed for preparing anticancer nanocrystals. In this review, preparation methods and in vivo performance of anticancer nanocrystals are discussed first, followed by an introduction of hybrid nanocrystals in cancer theranostics.

Keywords: biodistribution • cancer • formulation • nanocrystals • pharmacokinetics • theranostics

Today, cancer is one of the most life-threat-ening diseases. Globally, approximately 14.1 million new cancer cases occurred in 2012, resulting in about 8.2 million deaths [1]. WHO projects a 45% increase in cancer-related mortalities by 2030 [1]. New therapeutic approaches, including anticancer molecules and delivery strategies, are urgently needed, while current therapeutic options, by and large, fail to win the war against cancer. In this review, we will discuss the emerging drug nanocrystal formulation of anticancer agents, in particular, how nanocrystals are prepared and how they performance in vivo relatively to conventional delivery methods. Nanocrystals are regarded as crystalline particles of a drug whose sizes are generally smaller than 1 μm.

Despite oral medications being more accepted, intravenous injection is still the predominant route for cancer treatment [2]. This is largely due to the fact that many can-cer drugs are not readily absorbed following oral administration, due to poor solubility and/or digestive tract toxicity, limiting the oral absorption below therapeutic levels. This situation will not improve because more than 40% of newly synthesized drug mol-ecules in development are poorly water sol-

uble [3–6]. The proportion of water-insoluble drugs developed in oncology, anti-infective and antiviral treatment is even higher [7]. Given the increase in cancer morbidity and poor solubility of many drug molecules, the need for methods to effectively deliver anti-cancer treatment remains even imperative, particularly for intravenous administration.

Development of nanocrystals emerged amid various shortcomings of existing deliv-ery techniques for poorly soluble drugs. Organic solvents and surfactants are regu-larly used for solubilization, but many solu-bilizing agents raise safety concerns [8]. Hydroxypropyl-β-cyclodextrin and cremo-pher-EL have been utilized in commercial products, but their usage can lead to severe toxic side-effects, including hypersensitivity and nephrotoxicity [9].

A poorly soluble drug may be encapsu-lated and/or solubilized by polymers, lipids or surfactants into nanosized particles suit-able for intravenous administration [6]. Sev-eral nanoparticle-based delivery platforms, including liposomes, polymeric nanopar-ticles, solid lipid nanoparticles and micelles, have been developed for various anticancer agents [10]. Nonetheless, only a handful of anticancer drug products that are based on

Developing nanocrystals for cancer treatment

Yi Lu‡,1,2, Yan Chen‡,1,3, Richard A Gemeinhart4, Wei Wu2 & Tonglei Li*,1

1Department of Industrial & Physical

Pharmacy, College of Pharmacy, Purdue

University, West Lafayette, IN 47907,

USA 2Department of Pharmaceutics, School

of Pharmacy, Fudan University, Shanghai,

201203, China 3Department of Pharmaceutics, School

of Pharmacy, Second Military Medical

University, Shanghai, 200433, China 4Departments of Biopharmaceutical

Sciences, Bioengineering

& Ophthalmology & Visual Sciences,

The University of Illinois, Chicago,

IL 60612, USA

*Author for correspondence:

Tel.: +1 859 257 1472

[email protected] ‡Authors contributed equally

For reprint orders, please contact: [email protected]

rgemeinh

Rectangle

rgemeinh

Copyright

10.2217/NNM.15.73 Nanomedicine (Lond.) (Epub ahead of print) future science group

Review Lu, Chen, Gemeinhart, Wu & Li

nanoparticle delivery technologies have been approved by the US FDA, including Doxil® (doxorubicin in liposomes), DaunoXome® (daunorubicin in lipo-somes) and Abraxane® (paclitaxel nanoparticles). Plat-form instability, limited drug loading (i.e., percent of the drug amount loaded in a delivery system), high manufacturing cost, scale-up difficulties and quality control difficulties contribute to the limited acceptance of nanoparticle-based delivery systems into the clinic.

Drug nanocrystals, on the other hand, may be injected intravenously without resorting to any car-rier chemicals for encapsulation and/or solubilization, although surfactants or polymers are needed in most cases for stabilizing pure drug crystals to prevent or reduce their aggregations [3,11,12]. Nanocrystals are broadly recognized as drug crystals with a particle size ranging from dozens to a few hundreds of nanometers. Because size reduction to the nanometer range leads to increased dissolution rates [3,13], nanocrystal formu-lations initially found applications in oral administra-tion of poorly soluble drugs, improving the oral bio-availability, reducing pharmacokinetic variability in the fed and fasted states and variability among patient populations [14,15]. To date, commercial nanocrystal-based drug products are mostly for oral administration with one exception, for a product administered by the intramuscular route (Table 1).

Despite no approved products to date, nanocrys-tal formulation has gained considerable attention for intravenous delivery of anticancer drugs. This is due to distinctive characteristics of nanocrystals compared with other nanoparticle platforms [6,7,12]. Sans carrier chemicals, a nanocrystal formulation can not only eliminate any toxic side-effects inflicted by the encap-sulating/solubilizing excipients, but also offer a drug loading as high as 100%, resulting in satisfactory ther-apeutic concentrations at low dose [16]. Improved phar-macokinetics and biodistribution of anticancer drugs are expected because of the uniform and stable physical nature of crystalline particles [13,14]. Most importantly, physical instability issues inherent with other delivery systems are largely circumvented by the nanocrys-tal formulation [9,17,18]. Given the intrinsic solubility of many anticancer drugs in the practically insoluble range (<0.1 mg/ml) [19] combined with the high dose typically required for such treatment, nanocrystals are not expected to dissolve rapidly in the blood upon intra-venous administration mainly due to lack of local mix-ing and initially insufficient volume of distribution [14]. Limited interactions between nanocrystals and plasma proteins may prevent drug molecules from enzymatic degradation, further enhancing the safety and effi-cacy of anticancer agents [20]. In addition, nanocrys-tals injected into the bloodstream are sequestered by

mononuclear phagocytic cells, which in turn may act as drug depots to further improve pharmacokinetics and biodistribution of anticancer drugs [13,14].

Nanocrystals are produced by either top–down or bottom–up techniques [21]. The top–down approaches generally involve applying high-energy mechanical forces to comminute large crystals by media milling (NanoCrystals®) or high-pressure homogenization (HPH; IDD-P®, DissoCubes® and Nanopure®) [13,14]. Figure 1 illustrates the general principles of these techniques, which have been widely used to formu-late insoluble drugs, particularly in the production of orally administered products [15,22]. The HPH tech-nique forces larger drug crystals through an impact valve under fluidic pressure, causing the crystals to break into smaller pieces. Particle size is regulated by the pressure and the gap between the impact valves. The media milling, on the other hand, resorts to the grinding by hard particles, such as cerium, yttrium-stabilized zirconia, stainless steel and highly cross-linked polystyrene resin-coated beads [13], which are blended with larger drug crystals. Due to the grinding and attrition with milling particles, drug crystals are comminuted and nanocrystals can then be separated from the milling media. Particle size may be controlled by the material loading, amount and size of milling beads, and milling conditions [23]. However, contami-nation from the grinding media can lead to unex-pected side-effects. In addition, uniform size distribu-tion is difficult to achieve, causing particle aggregates and growth due to Ostwald Ripening. The poor size control is compounded further by decreased crystal-linity. Moreover, a top–down process is generally time consuming and energy intensive [24].

Conversely, bottom–up techniques grow nanosized crystals directly from drug solution. Several general bottom–up methods are illustrated in Figure 2. By mixing a drug solution with a solvent in which the drug does not dissolve well, in other words, antisolvent, the drop in solubility leads to nucleation and growth of drug crystals. When the nucleation is promoted abruptly, for example, by sonication and mechanical stirring, nanosized crystals may be obtained. The mix-ing of the antisolvent and drug solution can be done in a batch or continuous mode. When CO

2 is used as

a solvent at its critical state to dissolve or precipitate drug crystals, the crystallization process takes advan-tage of easy removal of the solvent. Compared with the top–down approach, the crystallization techniques have greater potentials to produce nanocrystals with superior crystallinity and of narrow particle size distri-bution and uniform morphology, in addition to mod-est energy requirement and fewer issues of contami-nation from the manufacturing equipment [6,22,25].

10.2217/NNM.15.73future science group

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10.2217/NNM.15.73 Nanomedicine (Lond.) (Epub ahead of print)

Figure 1. Schematic illustration of common top–down techniques in which coarse drug crystals are comminuted by high pressure or milling media. In the high-pressure homogenization technique, larger crystals are forced through a thin gap at an extremely high velocity and broken into smaller pieces by impaction or shear forces. Drug crystals are subjected to mechanical attrition by milling beads and broken up in the media milling technique.

Microcrystals NanocrystalsCoarse crystals

Size reduction

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future science group


Nanocrystals generated by the bottom–up technique are, thus, more suitable for intravenous injection of anticancer compounds. Technical challenges, how-ever, remain over the control of nucleation and crystal growth. Scale-up of nanocrystal production from solu-tion has proven to be difficult and inconsistant, result-ing in no marketed parenteral products. Still, various bottom–up techniques have been developed making their way into commercial production of nanocrystals, including hydrosols, high-gravity controlled precipi-tation technology (HGCP), confined impinging jet technology, supercritical fluid (SCF) technology and sonoprecipitation [26].

Pure nanocrystals are prone to aggregation due to high surface energy and high surface area [27]. Even when appropriately sized nanocrystals are formed, they tend to grow in size during production and storage. Stabilizing molecules such as surfactants are intro-duced in the preparation to yield stable nanocrystals and reduce surface energy, although some nanocrystal systems without stabilizers have been reported [28,29]. Stabilizers can adsorb onto the surface of nanocrystals and provide steric and/or electrostatic stabilization effects. Surface treatment can also be utilized to render nanocrystals with enhanced pharmacokinetic prop-erties. For example, biodistribution of nanocrystals may be further regulated by surface modification with hydrophilic materials to prolong the blood circula-tion; cancer-targeting ligands may be used to enhance drug accumulation in the tumor [30–32]. In addition, coated nanocrystals can reduce multidrug resistance (MDR) [33–35].

Because of the advantages of using nanocrystals over conventional delivery systems, an increasing number of anticancer drugs have been formulated into nanocrystals for intravenous delivery (Table 2) [30–49]. As expected for the array of agents examined, vari-ous types of cancers, including pancreatic, stomach, lung and prostate, have being studied with the treat-ment of nanocrystals. In addition, hybrid nanocrys-tals have been developed to achieve concurrent tumor therapy and imaging [50–52]. In the following sections, nanocrystals in development for cancer treatment is reviewed with a focus on their preparation methods and performance. Finally, hybrid nanocrystals will be discussed and their potential in the clinic explored.

Preparation of nanocrystal formulationTop–down processesAs previously mentioned, nanocrystals generated by the bottom–up technique should be more suitable for intravenous administration of anticancer compounds. A majority of anticancer nanocrystals reported in the literature, however, were prepared by the top–down means, among which, HPH remains the most popu-lar (Table 2). The general process of HPH consists of three steps [32,36,37,39,44,45]: dispersion of drug powder in water containing stabilizer; initial comminution of coarse drug particles with high-shear or low-pressure homogenization to prevent the homogenizer cham-bers from blocking; and homogenization under high pressure with repeated cycles to achieve the desirable particle size and size distribution. The homogeniza-tion pressure and numbers of cycles significantly affect

10.2217/NNM.15.73

Figure 2. Schematic illustration of common bottom–up techniques in which drug nanocrystals grow from solution. Crystallization can be achieved by mixing a drug solution with an antisolvent under sonication or high pressure for promoting nucleation. The crystallization process may also take advantage of easy evaporation of supercritical CO2. SCF: Supercritical fluid.

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future science group

Developing nanocrystals for cancer treatment Review

the particle size and size distribution of resultant drug crystals. It is reported that the particle size and poly-dispersity index (PDI) of amoitone B nanocrystals became gradually decreased with a stepwise increase in homogenization pressure or process cycles [39]. How-ever, continuous increase in pressure and/or process cycles has little effect on size reduction [39]. As drug particles become more uniform with reduction in interparticle aggregates, energy input exponentially increases to further break the crystals [11].

The second commonly used top–down technique is precipitation combined high-pressure homogenization (PCHPH) to prepare nanocrystals (Table 2). In the pro-cess of PCHPH, the drug substance first precipitates in a liquid medium, which is then used to replace the first two steps in HPH [30,43]. The technique requires that drugs can dissolve at least in one solvent, while a misci-ble nonsolvent should be available for the precipitation process. Thus, selection of proper solvents may present challenges as potential toxicities can further limit the solvent availability. Complete removal of solvents from the preparation can also be a daunting task [11].

Wet-ball milling (WBM) is the least used method to prepare anticancer nanocrystals (Table 2). It is sim-pler, directly comminuting drug suspensions with milling media reducing the particle size to a certain value. However, the process requires significant mill-ing durations, for example, in order to reach a desirable

particle size and PDI of an anticancer kinase inhibitor, MTKi-327, the milling lasted 66 h at room tempera-ture with 0.5 mm ZrO

2 as the milling media [40]. Pos-

sible contamination from milling media leads to safety concerns. All of these factors limit the application of WBM in the preparation of anticancer nanocrystals.

To control the particle size and distribution, chemi-cal stabilizers become indispensable in the particle comminution process (Table 2). Both the amount and the type of stabilizers affected the particle size and size distribution in the preparation of amoitone B nano-crystals by HPH [39]. Combined use of Pluronic® F68, lecithin and hydroxypropyl methyl cellulose (HPMC) showed better control on the particle properties than any single surfactant, likely due to the synergetic protection by electrostatic repulsion and steric hin-drance [53]. When the total amount of combined sta-bilizers was two-times that of amoitone B, the resul-tant particles became more uniform ranging between 200 and 300 nm, possibly as the result of stabilizers fully covering the surface of the nanocrystals. Because of toxic side effects, HPMC was nonetheless excluded from the final preparation of amoitone B nanocrystals. Similarly, studies demonstrated the combined use of lecithin, poloxamer, HPMC and poly(vinyl pyrrol-idone) (PVP) as stabilizers in the preparation of ric-cardin D [37] and puerarin [45] nanocrystals resulted in the greatest control of particle properties.


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h,

t 1/2β

0. 6

7 h

, t1

/2γ 8

.44

6 h

MTK

i-32

7W

BM

Plu

ron

ic F

108

, Li

po

id S

75 a

nd

g

luco

se

195

-20

Mo

use

Solu

tio

n: A

UC

760

± 1

90

ng

•ml-1

•h,

Cm

ax 9

40

± 3

93 n

g•m

l-1, t

1/2 5

.98

± 1

.84

h

[40]

Nan

ocr

ysta

ls: A

UC

178

5 ±

323

ng

•ml-

1 •h

, Cm

ax 5

076

± 1

709

ng

•ml-1

, t1

/2 5

.28

± 2

.02

h

† Relative efficiency, calculated by the area under the curve value of nanocrystals divided by that of the reference preparation.

Tab

le 2

. Pro

du

ctio

n m

eth

od

an

d p

har

mac

oki

net

ic p

rop

erti

es o

f in

trav

eno

usl

y ad

min

istr

ated

an

tica

nce

r n

ano

crys

tals

.



Dru

gM

eth

od

(s

cale

)St

abili

zer

Part

icle

siz

e (n

m)

Zeta

po

ten

tial

(m

v)A

nim

al

mo

del

Phar

mac

oki

net

ics

(PK

)B

iod

istr

ibu

tio

n (

BD

, re

c†)

Ref

.

CP

TPU

(1

mg

)

250

R

atSo

luti

on

: AU

C 2

104

.07

ng

·ml-1

·h,

MR

T 1.

39 h

, CL

755.

64

ml·

h-1,

t 1/2α 0

.11

h, t

1/2β2

.86

h

[41]

N

ano

crys

tals

: AU

C 8

29.7

9 n

g·m

l-1·h

, M

RT

2.83

h, C

L 18

96.

76 m

l·h

-1,

t 1/2α 0

.23

h, t

1/2β1

2.76

h

HC

PT

PU (

50

mg

)Fe

tal c

alf

seru

m16

8

H22

-tu

mo

r-

bea

rin

g

mo

use

B

loo

d 8

.78

, hea

rt 7

.01,

live

r 41

0.49

, sp

leen

46.

05, l

un

g

40.

63, k

idn

ey 4

2.03

, tu

mo

r 5.

72

[42]

HC

PT

PCH

PH(4

0 m

g)

Lip

oid

S75

an

d

Plu

ron

ic F

68

287

-32

[43]

SBH

PH(1

%)

Leci

thin

an

d

Plu

ron

ic F

68

Smal

ler:

127

B

igg

er: 6

42

[44]

PRH

PH(1

%)

Leci

thin

an

d

HPM

C4

82

[45]

HPP

HA

P(3

mM

)

110

-40

[46]

PT

XPU (3

mg

)Tr

f 30

3-1

3.6

Mo

use

[47]

PIK

-75

HPH

(30

0 m

g)

Plu

ron

ic F

68

(no

nta

rget

ed)

/Plu

ron

ic

F68

-fo

lic a

cid

(t

arg

eted

),

leci

thin

No

nta

rget

ed:

182

Targ

eted

:16

1

No

nta

rget

ed:

-39

Targ

eted

:-3

4

SKO

V-3

tu

mo

r b

eari

ng

m

ou

se

Susp

ensi

on

: AU

C 1

.09 μg

·ml-1

·h, C

ma

x 0.

80 μg

·ml-1

No

nta

rget

ed: A

UC

1.4

6 μg

·ml-1

·h, C

ma

x 0.

59 μ

g·m

l-1

Targ

eted

: AU

C 6

.74 μg

·ml-1

·h, C

ma

x 2.3

8 μg

·ml-1

No

nta

rget

ed: t

um

or

4.6

9,

liver

0.7

7, k

idn

ey 0

.72,

sp

leen

13.

76, l

un

gs

0.91

Targ

eted

: tu

mo

r 8

.90,

live

r 0.

80,

kid

ney

0.3

8, s

ple

en

15.9

0, lu

ng

s 0.

82

[32]


Tab

le 2

. Pro

du

ctio

n m

eth

od

an

d p

har

mac

oki

net

ic p

rop

erti

es o

f in

trav

eno

usl

y ad

min

istr

ated

an

tica

nce

r n

ano

crys

tals

(co

nt.

).



Dru

gM

eth

od

(s

cale

)St

abili

zer

Part

icle

siz

e (n

m)

Zeta

po

ten

tial

(m

v)A

nim

al

mo

del

Phar

mac

oki

net

ics

(PK

)B

iod

istr

ibu

tio

n (

BD

, re

c†)

Ref

.

DT

XH

PH(0

.1%

)Le

cith

in/

DSP

E-PE

G20

00

(pLN

S);

pLN

S: 2

04

tLN

S: 2

21p

LNS:

-3

4tL

NS:

-28

B16

tu

mo

r-b

eari

ng

m

ou

se

Du

op

afei

®: C

ma

x 481

.62 μg

/ml;

t 1/2α0.

31

h; t

1/2β

1.78

h; A

UC

30

8.4

2 m

g·l

-1·h

; M

RT

1.76

h; C

L 0.

195

l·h

-1·k

g-1

pLN

S: h

eart

0.9

7, li

ver

17.8

6, s

ple

en 9

.80,

lun

g

3.23

, kid

ney

0.9

3, t

um

or

5.22

[31]

Le

cith

in/D

SPE-

PEG

200

0/

DSP

E-PE

G20

00

-FA

(t

LNS)

p

LNS:

Cm

ax 2

17.9

2 μg

/ml;

t 1/2α0.

230

h;

t 1/2β3

.68

h; A

UC

489

.65

mg

·l-1·h

; MR

T 4

.229

h; C

L 0.

100

l·h

-1·k

g-1

tLN

S: h

eart

0.9

6, l

iver

17

.14

, sp

leen

9.9

7, lu

ng

3.

37, k

idn

ey 0

.95,

tu

mo

r 5.

92

tLN

S: C

ma

x 213

.45 μg

/ml;

t 1/2α0.

230

h;

t 1/2β3

.70

h; A

UC

511

.30

mg

·l-1·h

; MR

T 4

.230

h; C

L 0.

097

l·h

-1·k

g-1

PT

X3P

NE

T(0

.5 m

g)

Plu

ron

ic F

127,

Pl

uro

nic

F12

7-fo

lic a

cid

122

+1

[48]

PT

XPC

HPH

DO

TAP

160

–170

+13

[30]

PT

X3P

NE

T(0

.5 m

g)

TPG

S15

0

[35]

PT

XEP

AS

(0.2

g)

TPG

S13

5

Mo

use

Solu

tio

n: A

UC

11.4

0 m

g·l

-1·h

, MR

T 6.

02

h, C

L 0.

88

l·h

-1·k

g-1, t

1/24

.17

h, C

ma

x 9.8

7 m

g·l

-1

[33,34

]

N

ano

crys

tals

: AU

C13

.15

mg

·l-1·h

, MR

T 14

.79

h, C

L 0.

66 l

·h-1·k

g-1, t

1/210

.25

h,

Cm

ax6

.45

mg

·l-1


Tab

le 2

. Pro

du

ctio

n m

eth

od

an

d p

har

mac

oki

net

ic p

rop

erti

es o

f in

trav

eno

usl

y ad

min

istr

ated

an

tica

nce

r n

ano

crys

tals

(co

nt.

).



Bottom–up processesAs shown in Table 2, the bottom–up approach plays a less significant role in preparing anticancer nanocrys-tals to date. Nanocrystals obtained from bottom–up processes bear smaller particle sizes than those by particle milling or homogenization techniques. The production scale is nonetheless smaller. In some cases, nanocrystals can be produced without using any sta-bilizers. Consequently, drug loading becomes signifi-cantly higher. The essence of a bottom–up process is precipitation of dissolved drug molecules, which consists of two crucial steps, nucleation and subse-quent crystal growth to generate small and uniform nanocrystals. Therefore, the nucleation step needs to be carefully controlled [6]. The more nuclei formed in the nucleation stage, the less growth of each nucleus in the end.

Cavitation effects resulting from ultrasonic waves can effectively promote rapid and thorough nucle-ation [6]. Ultrasound can further minimize particle agglomeration by breaking particle contacts [54]. As such, precipitation-ultrasonication (PU) has become the mostly used bottom–up method (Table 2). The process starts with drug substances dissolved in a sol-vent and subsequently injected into a miscible anti-solvent (usually water) under vigorous stirring and intense sonication [41,42,47]. Production factors affect-ing the particle size and distribution by PU were inves-tigated in one study of preparation of paclitaxel (PTX) nanocrystals [47]. Good solvents including methylene chloride, ethyl acetate and dimethyl sulfoxide pro-duced significantly larger nanocrystals compared with methanol and ethanol; the difference was attributed to the diffusion rates of the solutions dissipating in the antisolvent, water. Slow diffusion impeded forma-tion of supersatured domains when mixing, reducing nucleation rates and thus leading to larger crystals [22]. Additionally, for a given amount of PTX, increasing the antisolvent/solvent ratio enhanced the degree of supersaturation and promoted nucleation, forming smaller nanocrystals. When the PTX concentration exceeded a certain value, however, the nanocrystal size increased dramatically. Similar observations were also made in the preparation of beclomethasone dipro-pionate nanocrystals by the antisolvent precipitation (AP) [55]. Although high supersaturation is desired for promoting nucleation, a larger number of nuclei may increase collisions between solid particles and lead to particle aggregates. Using stabilizing chemicals can affect the particle size as well. In most cases, nonethe-less, the presence of stabilizers caused the nanocrystal size to increase, possibly due to decreased supersatura-tion as a result of solubilization by the stabilizers [47]. Thus, the timing of the introduction of the surfactant

to the crystallization media is critical, with the addi-tion of surfactant being best after nanocrystals are initially formed.

Evaporative precipitation into aqueous solution (EPAS) is another bottom–up method that has been used to prepare anticancer nanocrystals [33,37]. In this process, drugs are generally dissolved in 60°C ethanol and then added dropwise into 0°C water under rapid stirring. The temperature difference between the good solvent and antisolvent further enhances supersatura-tion and hence promotes nucleation. Nonetheless, the dropwise addition may not benefit for controlling the crystal size, because the newly added drug molecules preferentially grow onto the existed nuclei, leading to larger crystals. Still, a smaller particle size was obtained by EPAS (184 nm) than by HPH (815 nm) in the preparation of riccardin D nanocrystals [37], indicating great control of particle size can be achieved with this method.

Three-phase nanoparticle engineering technology (3PNET) involves three steps in preparing nanocrys-tals: precipitation of amorphous PTX; hydration of the precipitates; and sonication of the hydrated suspensions to form nanocrystals. Although PTX can form amor-phous solid, stabilizers are required in this process to prevent PTX crystallization in step 2 and form amor-phous aggregates. Then crystals grow from the amor-phous state upon sonication. Using this technique, stabilized nanocrystals were formed by surface coating with stabilizers [35,48]. Both Pluronic F127 and d-α-tocopheryl PEG 1000 succinate (TPGS) have been successfully used as stabilizers to prepare PTX nano-crystals with particle sizes around 150 nm (Table 2). However, in order to form amorphous precipitate in the first step, at least four- and onefold amount of F127 and TPGS compared with PTX was needed, respec-tively [35,48]. Lower drug loading and possible toxicity resulted from large amount of stabilizers might thus limit the development of this technique.

Surface treatmentWhether a top–down or bottom–up approach is used, stabilizing chemicals are often used to treat the surface of nanocrystals. Usually, surfactants adsorb onto the surface of nanocrystals through physical interactions. Not only can adsorption of surfactants control the par-ticle size of nanocrystals, but also prevent nanocrystals from aggregating. In addition, with a specific design of coating materials, surface treatment can further endow nanocrystals with additional functions such as active tumor targeting [30–32,48] or anti-MDR [33,35].

Serum proteins have been investigated for enhanc-ing the stability of PTX nanocrystals during post-processing steps, including centrifugation, filtration



and freeze-drying [47]. It was found that the average particle size of PTX nanocrystals increased by 115 ± 10% after centrifugation and drying. After surface coating with human serum albumin (HSA) or trans-ferrin (Trf), the increase in particle size was hindered during subsequent processing steps. Trf showed a superior stabilizing effect to HSA with particle size increased by only 5.57 ± 2.7% after centrifugation and drying [47]. Adsorption of HSA or Trf molecules onto the surface of PTX nanocrystals provided a physical barrier that prevented nanocrystals from aggregation. On the contrary, surface treatment with IgG caused the increase of particle size of PTX nanocrystals, pos-sibly resulting from aggregation of the coated IgG proteins. Additionally, Trf treatment showed higher in vivo tumor inhibition rate, 45.1%, compared with 28.8% for uncoated nanocrystals. Yet, no difference in in vitro cellular uptake was observed between Trf-treated and untreated nanocrystals, suggesting that the mechanisms of improved activity may not isolated to improved cellular uptake [47].

Surface treatment of nanocrystals with ligand-conjugated polymers have been desired to engage the receptors on cancer cell membranes for enhanc-ing cellular uptake and elevating intracellular drug concentrations [31,32,48]. In one study, folic acid (FA) was introduced to PTX nanocrystals (FA-F127-NCs) by stabilizing the drug particles with FA-conjugated Pluronic F127 using 3PNET [48]. FA-F127-NCs exhibited a greater anticancer activity (p < 0.001) compared with untreated nanocrystals when cocul-tured with KB cells, known to overexpress folate receptors. Similarly, nanocrystals of PIK-75, a phos-phatidylinositol 3-kinase inhibitor, were prepared by HPH with FA-Pluronic F68 conjugates as the sta-bilizer (FA-F68-NCs) [32]. In vitro studies showed that FA-F68-NCs were taken up by SKOV-3 cells, a folate receptor-positive ovarian cancer cell line, twice as much as untreated nanocrystals. The augmented uptake resulted in a reduced IC

50 for the FA-F68-NCs

relative to the untreated nanocrystals (54 vs 152 nM). In another study, PEG-modified (pLNS) and FA-modified (tLNS) docetaxel nanocrystals were pre-pared by HPH and stabilized with 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE)-PEG2000 and DSPE-PEG2000-FA, respectively [31]. The cyto-toxicity of tLNS against B16 (folate receptor positive) cell lines was more pronounced than pLNS, while there was no significant difference between pLNS and tLNS against HepG2 (folate receptor negative). These results suggest that surface treating of drug nanocrys-tals with ligands may enhance internalization by tar-geted cancer cells and possibly lead to increase in the local drug concentration in vivo.

One interesting study utilized polycations to coat PTX nanocrystals aiming to facilitate nonspe-cific cellular uptake of the nanocrystals [30]. IgG, protamine or 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) were selected for coating the PTX nanocrystals during the postproduction steps of centrifugation and redispersion. DOTAP-coated nanocrystals (DOTAP-NCs) showed faster cellular uptake than IgG-, protamine-coated and uncoated nanocrystals by macrophages, especially by immature CD14+CD16- cells relative to mature CD14+CD16+ cells. Additionally, DOTAP-coated nanocrystals were taken up more readily by peritoneal macrophages and OVCAR-3 cancer cells than the uncoated nanocrys-tals. Subsequent in vivo experiments demonstrated an extended release for the DOTAP-coated nanocrystals up to 30 days following both the subcutaneous and intraperitoneal routes of administration. Further-more, the subcutaneously administrated DOTAP-coated nanocrystals were as effective as five cycles of intravenous Taxol treatment in an orthotopic mouse model of head and neck cancer. A combination therapy of intravenous Taxol plus intraperitoneally DOTAP-coated nanocrystals effected significantly stronger responses than intravenous Taxol alone in OVCAR-3 bearing mice. The results suggest that DOTAP-coated nanocrystals could be trafficked to the tumor site more preferentially by macrophages, especially CD14+CD16- monocytes cells bearing CCR2 tumor targeting capability [30].

Surface treatment of PTX nanocrystals with TPGS, a P-gp inhibitor, was demonstrated to overcome the MDR by cancer cells [33–35]. The nanocrystals (TPGS-NCs) were prepared by both 3PNET [35] or EPAS tech-nology [33]. The TPGS coating rendered the crystals reduced dissolution and improved stability. Tested in a resistant human ovarian cancer cell line (NCI/ADR-RES), TPGS coated nanocrystals significantly increased the apoptotic response up to more than 95%, relative to 19.4% when cells were treated with Taxol [35]. Treatment by TPGS-nanocrystals resulted in an IC

50

of approximately 5 μM while no effect was registered with untreated crystals. In vivo studies further demon-strated apparent tumor regression by the TPGS-nano-crystals in a NCI/ADR-RES xenograft murine model, while that equal dosage of Taxol treatment ineffectively inhibited the tumor growth. Similarly, reduced drug resistance in P-gp overexpressing H460 human lung cancer cells was observed both in vitro and in vivo with TPGS-nanocrystals [34]. However, mixed solutions of TPGS and PTX could not achieve the same tumor inhibit effects as TPGS-nonocrystals. It is thought that the TPGS coating enabled enhanced the local concen-tration of the drug in the tumor site and effectively



impeded the P-gp efflux function contributing to the improved therapeutic effect [56].

Zeta potential is an important surface property of nanoparticles often used to evaluate the potential of particle aggregation and colloidal stability. A large value (negative or positive) enables particles to resist aggregation; when the value is small, attractive (van der Waals) forces may overcome the (electrostatic) repul-sion leading to flocculation and clustering of the par-ticles. The zeta potential values listed in Table 2 mostly ranged between -20 and -30 mV, suggesting suitable stability. Noted that, in addition to zeta potential, the colloidal stability also depends on particle properties, solution chemistry and use of surfactants.

Pharmacokinetics & biodistributionPharmacokineticsInjected intravenously, nanocrystals are generally rec-ognized as exogenous entities and taken up by the immune system [57]. In one study, crystalline particles were observed ex vivo in macrophages by transmission electron micrography of a rat spleen following intra-venous injection of itraconazole nanocrystals [58]. Due to slow dissolution from the phagolysosomes and sub-sequent release from the phagocytic cells, the pharma-cokinetic behaviors of nanocrystals exhibits a marked difference from those of the solution formulation [59]. This leads to prolonged mean residence time (MRT, the average time of a drug residing in the body), half-life (t

1/2, the time to reduce 50% of the plasma drug

concentration, which is further divided into distribu-tion half-life, t

1/2α, and elimination half-life, t1/2β, to

specify the reduction cause by either distribution or metabolism), increased volume of distribution (V

d,

the apparent volume in which a drug is distributed in the body to convert the plasma drug concentration to the drug amount), and reduced maximum serum con-centration (C

max), compared with the solution. Such

pharmacokinetic characteristics have been observed for most intravenous administrated anticancer nano-crystals (Table 2). While mice are used for determining drug biodistribution, the pharmacokinetic parameters are usually obtained from rats and rabbits because of their larger blood volume.

Particle size has shown a great influence on the pharmacokinetics of drug nanocrystals [36,37]. Ricca-rdin D nanocrystals, prepared both by a bottom–up and a top–down method, achieved the mean particle size of 184 and 815 nm, respectively. Upon intravenous administration to rats, the smaller nanocrystals exhib-ited similar area under curve (AUC, the integral of a drug concentration-time profile as an indicator of the total drug exposure over time), MRT, clearance (CL, the plasma volume cleared of the contained drug per

unit time) and the time to reach Cmax

(tmax

), to that of the riccardin D solution, except a C

max 50% higher was

acheived. The larger nanocrystals, on the other hand, led to the increase in AUC (2.65-fold higher), MRT (1.96-fold higher) and C

max (5.61-fold higher) relative

to that of either the smaller nanocrystals or the solu-tion. Similarly, in a rabbit model, smaller oridonin nanocrystals (103 nm) possessed a drug concentra-tion–time curve similar to that of the drug solution, while the larger crystals (897 nm) showed a markedly delayed clearance from blood [36]. These results seem to indicate that, at least in the animal models tested, larger nanocrystals enable higher bioavailability and extended clearance, compared with smaller crystals. It is hypothesized that the smaller nanocrystals dis-solve relatively quickly in blood, minimizing phago-cytosis by the reticuloendothelial system (RES), and thus bear similar in vivo behaviour to the solution [59]. On the contrary, larger particles are more likely phago-cytosed by the RES and preserved as drug depots, allowing subsequent release of some free drug from the macrophages and yielding delayed pharmacokinetic characteristics [36,37].

Plasma pharmacokinetics of nanocrystals do not necessarily correlate with organ and tumor biodistribu-tion [41]. As examined in a lung metastatic breast can-cer murine model, camptothecin nanocrystals achieved a 90% reduction in tumor metastasis, compared with 55% reduction observed by the solution formulation. This anticancer effectiveness was not corroborated by observed AUC values (2104.1 h·ng·ml-1 for solution vs 829.8 h·ng·ml-1 for nanocrystals), but by MRT (1.39 h for solution vs 2.83 h for nanocrystals), further sug-gesting that the biodistribution of nanocrystals in other organs and tumors may be more important than the plasma concentration and pharmacokinetics.

BiodistributionBecause of the uptake and subsequent transport by phagocytic cells following intravenous injection, nano-crystals distribute differently to tissues and organs relative to administered solution [57]. Compared with the solution (Table 2), enhanced accumulations of nanocrystals were detected in macrophage-abundant organs, including liver, spleen, lung and kidney. In particular, the AUCs of hydroxycamptothecin nano-crystals in liver, spleen, lung and kidney were measured 410-, 46-, 40- and 42-times the organ-specific AUCs obtained following intravenous administration of drug solution at the same dose, respectively (Table 2). Simi-larly, in one of our previous studies, the AUC

0−24 of

intravenous administered camptothecin nanocrystals was 7.5-times higher in the lung than that of the salt solution investigated in a murine tumor model [60].



As discussed earlier, pharmacokinetics of riccardin D nanocrystals was generally influenced by nano-crystal size. Not surprisingly, larger nanocrystals were taken up more by the RES than the drug solu-tion, leading to greater accumulations to liver, spleen and lung [37]. Similar distribution characteristics were observed in the intravenous administration of oridonin nanocrystals [36]. Beyond just size, particle morphology also influences the biodistribution of nanocrystals. In one study, rod- and sphere-shaped 10-hydroxycampto-thecin nanocrystals were prepared with similar overall size and surface charge [61]. The rod-shaped nanocrys-tals effected more significant cellular uptake, in vitro cell growth inhibition and in vivo antitumor efficiency than the spherical nanocrystals. A previous study in our laboratory showed similar effects that needle-shaped camptothecin nanocrystals (ca. 250 nm) enabled more tumor accumulation in the lung, attributed to the high aspect ratio of the particles that make the nanocrystals difficult to escape following local entrapment [60].

Surface treatment of nanocrystals may modify their biodistribution behavior [62,63]. FA surface-treated PIK-75 nanocrystals increased AUC 1.90-fold at the tumor site compared with nonmodified nanocrystals, while similar AUC values in liver, spleen and lung were observed by these two nanocrystals (Table 2) [32]. FA, conjugated as DSPE-PEG2000-FA, was used to coat docetaxel nanocrystals, which demonstrated compa-rable in vivo distribution to DSPE-PEG2000-coated nanocrystals. Both surface-treated nanocrystals accu-mulated in the tumor more significantly than docetaxel solution (Table 2) [31]. These studies support the notion that nanocarriers can take advantage of the enhanced permeation and retention (EPR) effect, and active tar-geting by surface-bond ligands can only facilitate drug uptake after the nanocrystals reach to a tumor site [64].

Hybrid nanocrystalsGuest inclusion in a host crystal is a well-known phe-nomenon in solid-state chemistry, exemplified by col-ored diamonds in which impurity elements are embed-ded or grown into the lattice of the carbon crystal as defects. Inspired by the dyeing crystals [65], our labora-tory has developed drug nanocrystals that physically integrate fluorescent dyes by a bottom–up crystalliza-tion method [50–52]. Because of the physical integration of imaging agents with anticancer drugs as the hosting nanocrystals, concurrent tumor therapy and bio-imag-ing becomes feasible. The preparation method typi-cally starts with water-soluble fluorescent dyes, such as fluorescein, rhodamine B and FPR-749, dissolved in water (antisolvent), followed by mixing with a drug solution in a process similar to the PU method. The morphology of the hybrid nanocrystals are not altered

by the guest inclusion. Any surface-bond hydrophilic dyes are removed by rinsing the nanocrystals with water. Nanocrystals indeed become fluorescent due to the dye molecules trapped in the crystals [52]. The amount of integrated dyes is typical minimal (1% or less), effecting little impact on crystal properties of the host drug.

To investigate the potential use of hybrid nanocrys-tals for in vivo disease imaging, hybrid PTX nanocrys-tals with dual fluorescent probes were prepared with bioactivatable MMPSense® 750 and near infrared Flamma Fluor® FPR-648 fluorophores [50]. MMPSense 750 activated by matrix metalloproteinases (MMP), specifically MMP2 and MMP9 known to overexpress in many types of cancer, allowing the local proteolytic activity to be imaged. Tested in a murine model, the MMPSense was found within the tumor, while the nonspecific fluorophore, FPR-648, primarily resided in the liver. Fluorescent signal of MMPSense 750 diminished after day 3 while FPR-648 sustained for more than 7 days. The study demonstrated a possibil-ity of using hybrid nanocrystals to achieve anticancer therapy while monitoring the treatment of disease progression and drug distribution.

To further evaluate the theranostic potential, PTX nanocrystals were radiolabeled by physically integrat-ing 3H-PTX in the crystal, in addition to a fluorophore probe (FPI-749) that was also integrated for bio-imag-ing [51]. Tested in tumor-bearing mice, 3H-Taxol in a control group showed extensive distribution to major organs, including the heart, while the nanocrystals were rapidly cleared from the blood circulation by macrophages with more than 40% of the injected dose found in the liver. Furthermore, less than 1% of the injected dose of either Taxol or PTX nanocrystals were found in the tumor at any given time point. The drug concentration in the tumor when the nanocrystals were administered was nonetheless maintained from 8 to 72 h while it dropped for the Taxol solution. It is believed that release of 3H-PTX from engulfed nano-crystals in macrophages may be responsible for the prolonged retention. However, the biodistribution of the drug was not quantitatively correlative with the fluorescent imaging results. The drug accumulation in the tumor was less than 1% of the total injected dose, while the imaging data demarcated the tumor being strongly fluorescent. Discrepancies might be ascribed to the limitation of light penetration by the imaging system, as well as the variation in the pharmacokinetic properties of the drug molecules compared with the dye. It might also be possible that the hybrid nano-crystals preferentially accumulated at the periphery of the tumor. Using optical imaging for the indication of drug distribution needs to be carefully discerned, espe-



cially when imaging agents are physically integrated with the drug. Adopting new imaging modalities, such as fluorescence lifetime [66] and photoacoustic tech-niques [67,68], may further the application of hybrid nanocrystals for concurrent anticancer therapy and bioimaging.

ConclusionTo date, nanocrystal formulations have been predomi-nantly developed for enhancing the dissolution of orally delivered poorly soluble drugs for oral delivery. Nanocrystals have recently been explored as a viable delivery platform for intravenously administration of anticancer drugs. Compared with traditional antican-cer formulations, the physical stability, high drug load-ing and relative ease of production enable nanocrystal-based systems have the potential for being attractive alternatives for delivering poorly soluble anticancer drugs. Both top–down and bottom–up techniques have been advanced for preparing drug nanocrystals. While the bottom–up technique may be capable of better controlling the particle properties, the top–down approach dominates the commercial production of anticancer drug nanocrystals due to relative simplic-ity for scaling-up the process. Detailed understanding and control of the crystallization processes are nec-essary for achieving drug nanocrystals with desired particle properties, including size, size distribution and morphology. In addition, because stabilizing sur-factants are often used for maintaining nanocrystal uniformity, development and testing of effective stabi-lizers for intravenously injection remain an active area of investment for nanocrystal development, such as blood-derived and food-derived proteins [69–72].

Nanocrystal size has been shown to be critical for their in vivo performance. Smaller nanocrystals result

in faster dissolution than the larger ones. On the other hand, larger nanocrystals may enable extended release in the system resulting in greater drug accumula-tion at the tumor site, likely due to the fact that these crystals end up recognized by the RES and behave as drug depots, potentially within the RES cells. Surface treatment of nanocrystals with biocompatible and/or ligand-conjugated surfactants can further improve or enhance the anticancer treatment.

In conclusion, nanocrystals have been actively explored in anticancer drug delivery. Improved phar-macokinetics and biodistribution are achieved by nanocrystals compared with conventional formula-tions. Nanocrystals can be further treated with sur-factant chemicals for future improvement. Concur-rent anticancer treatment and bio-imaging become feasible with hybrid nanocrystals. Given the active efforts in the field, commercial products of anticancer nanocrystals will likely emerge.

Future perspectiveIt is interesting to note from Table 1 that no new nanocrystal-based drug products have been approved since 2009 despite a few being tested in clinical trials. Through various in vitro and in vivo studies, nano-crystal formulations have demonstrated their suit-ability and even superiority to conventional delivery systems for cancer therapy. One clear bottleneck in commercializing nanocrystal products for delivering anticancer compounds stems from enabling tech-niques for producing uniform and high-quality drug nanocrystals at the industrial scale. For this, bot-tom–up crystallization approaches bear significant promises and, we believe, breakthroughs in develop-ing novel methods and devices will occur in the near future.

Executive summary

Nanocrystals for cancer treatment• Nanocrystals hold advantages over conventional drug carriers for cancer treatment:

–Drug loading of nanocrystals can be as high as 100%; –Nanocrystals are free of organic solvents or other solubilizing chemicals; –The stability of nanocrystals is superior; –Nanocrystals protect drug molecules from enzymatic degradation.

• Surface treatment enables nanocrystals with improved stability and enhanced bioperformance. Long circulating, active targeting and antimultidrug resistance are few examples with surface-treated nanocrystals.

Improved pharmacokinetics & biodistribution• Sustained release of drug molecules from nanocrystals combined with the release from ingested nanocrystals

by mononuclear phagocytic cells can significantly improve the pharmacokinetic behaviors of anticancer drugs, leading to greater treatment efficiency.

• Much less toxic effects are expected from nanocrystals.Hybrid nanocrystals• Bioimaging agents can be physically embedded into the crystal lattice of drug nanocrystals.• Concurrent tumor treatment and bioimaging become feasible using hybrid nanocrystals.



Financial & competing interests disclosureThe authors acknowledge the support from Eli Lilly Seed Grant

and Chao Endowment. Y Lu thanks the financial support from

Chinese Scholarship Council for visiting at Purdue University.

The authors have no other relevant affiliations or financial in-

volvement with any organization or entity with a financial inter-

est in or financial conflict with the subject matter or materials

discussed in the manuscript apart from those disclosed.

No writing assistance was utilized in the production of this

manuscript.

ReferencesPapers of special note have been highlighted as: • of interest; •• of considerable interest

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Developing nanocrystals for cancer treatment...Drug nanocrystals, on the other hand, may be injected intravenously without resorting to any car-rier chemicals for encapsulation and/or