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Current Nanomedicine, 2020, 10, 1-23 1

REVIEW ARTICLE

Authors Review on Drug Nanocrystals: A Progress to Targeted

Delivery
Manish Kumar1,*, Nithya Shanthi3, P.S. Rajnikanth2 and Arun Kumar Mahato3

1
Department of Pharmaceutical Engineering and Technology, Indian Institute of Technology (BHU), Vara-
nasi, Uttar Pradesh (India), India; 2Department of Pharmaceutical Sciences, Babasaheb Bhimrao Ambedkar
University (A Central University), Lucknow, Uttar Pradesh (India); 3Department of Pharmaceutical Scienc-
es, Sardar Bhagwan Singh Post Graduate Institute of Biomedical Science and Research, Balawala, Deh-
radun, Uttarakhand (India)

ARTICLE HISTORY
Received: October 04, 2019
Revised: November 05, 2019
Accepted: November 14, 2019
DOI:
10.2174/2468187310666200221103827
Keywords: Modified nanocrystals, targeted delivery, surface grafting, 3rd generation nanocrystals.
Abstract: In the last few decades, researchers and pharmaceutical industries have been
developing new approaches to overcome the solubility and bioavailability limits observed
with poorly soluble drugs. With the advancement of nanotechnology, nanocrystals have
emerged as a great potential to overcome these limitations. Nanocrystals owing to its abil-
ity to modify the physicochemical and biological properties of the drug have gained wide-
spread attention among the research scientists. This review provides comprehensive detail
on the associated advantages, challenges, factors affecting physicochemical properties,
and optimization parameters about the stability of nanocrystals. In this review, the evolu-
tion of nanocrystals is discussed as first-generation simple nanocrystals, second-
generation nanocrystals within a carrier, and third-generation surface-modified nanocrys-
tals. It also provides a detailed account of various preparation methods and evaluation of
surface-modified nanocrystals. In the proposed "King Design," nanocrystals of the third
generation are placed on the top due to their advantage over other nanocarriers like high
drug payload, site-specific delivery, improved activity, commercial manufacturing, and
easy scale-up. Third generations nanocrystals can provide a novel therapeutic solution for
the site-specific, targeted, and efficient delivery for treatment of various acute as well as
chronic diseases with high stability and scale-up potential.

1. INTRODUCTION factor [1]. According to the Developability Clas-

In recent years, most of the newly discovered
compounds have poor aqueous solubility responsi-
ble for low bioavailability, unpredictable absorp-
tion, and lower therapeutic effect. Solubility is an
intrinsic property that determines the absorption
rate for poorly aqueous soluble drug belonging to
the Biopharmaceutical classification system (BCS)
Class II and IV and thus acts as a rate-limiting
*Address correspondence to this author at the Department of
Pharmaceutical Engineering and Technology, Indian Insti-
tute of Technology (BHU), Varanasi, Uttar Pradesh (India),
India; Tel: 7579112233;
E-mail: manishkumar.rs.phe19@itbhu.ac.in
sification System (DCS), drugs with too low in-
trinsic solubility for achieving the desired flux can
be further subdivided into dissolution limited
(Class IIa) and solubility limited (Class IIb) drug
also referred as brick dust model (insoluble in li-
pid, organic and aqueous solvent) and grease ball
model (only lipid-soluble) respectively [2, 3].
Several formulation strategies have emerged to
improve the solubility and subsequent bioavaila-
bility of drugs which included various nano-
particulate systems like nanofibers [4], quantum
dots [5], nanoclusters [6], nanorods [7], DNA
nano-carriers [8], liposomes [9], nanotubes [10],
micelles [11], dendrimer [12], SNEDDS [13] and

2468-1873/20 $65.00+.00 © 2020 Bentham Science Publishers

2 Current Nanomedicine, 2020, Vol. 10, No. 1
so on. These systems were based on some synthet-
ic polymers, natural polymers, or metallic com-
pounds like gold, silver, iron oxide, metal molly-
bdate, etc. [14]. Some features related to nano-
carriers are; superior characteristics reduced dose
and dosing frequency, and retarded drug rejection,
which provided an efficient, economical, flexible,
and targetable dosage form [15]. Though many
nanoparticle systems have been reported world-
wide for drug delivery, important aspects like low-
er drug payload, circulation instability, complicat-
ed manufacturing, and premature drug release are
their common drawbacks [16]. Recently, nano-
crystals have gained increasing interest and found
to be superior over other nano-particulate systems
due to high drug payload capacity, use of a minute
quantity of excipients, higher chemical stability,
lower toxicity, easy scale-up, and manufacturing
[3]. It also holds an advantage over various other
dosage forms like self-emulsifiers, cyclodextrin
based inclusions, solid dispersions, and solid-lipid
nanoparticles as it overcomes tedious preparation
methods and the excipient based toxicity [17].
Nanocrystals came in reckoning around the end of
the 20th century [18]. As reported by Chem M.L.
et al., 2017, about 82 submissions related to nano-
crystals were filed up until 2015 for various ail-
ments like cancer, infectious disease, inflammato-
ry disorders, etc. [19].
Nanocrystals are nanosized colloidal systems,
considered as pure drug crystals containing a very
less quantity of stabilizer with maximum drug load
(up to 100%) [20-22]. Nanocrystals modify the
intrinsic properties of raw material and drug, like
solubility and dissolution velocity, which leads to
supersaturation state of the drug instead of attain-
ing only thermodynamic solubility [2]. Increased
dissolution velocity can also be explained by the
Noyes-Whitney equation according to which de-
creasing size to nano-range is responsible for in-
creased surface area and thus dissolution. Fur-
thermore, amorphous nanocrystals present higher
dissolution being an advantage over crystalline
form [20]. Nanocrystals based formulation can be
administered via different routes and found to have
numerous advantages like the prolonged drug re-
lease, negligible side effects associated with stabi-
lizer used and no precipitation risk upon admin-
istration [19]. Nanocrystals have emerged as a
great potential for smooth and efficient delivery of
poorly soluble drugs with increased bioavailabil-
ity. Targeted or designer nanocrystals, capable of
Kumar et al.
controlling and preserving crystals morphology
can be a valuable tool for the delivery of poorly
soluble drugs [18].
At present, the growing interest of the pharma-
ceutical industry and academia in this technology
is quite evident by the continuous rise in the num-
ber of scientific research work and patents related
to drug nanocrystals, published in recent years.
Nanocrystals sprang up as a new technique with
multiple advantages and wide applications in vari-
ous fields; packaging of food products by use of
cellulose nanocrystals [23], stabilization of picker-
ing emulsions by use of chitin nanocrystals [24],
catalysing renewable energy reactions like hydro-
gen evolution, CO2 reduction, oxygen reduction
and evolution reactions [25], thermochromic ap-
plications (26), improving electrocatalytic proper-
ties [27], bioimaging [28], preparation of sens-
ing/fluorescent nanomaterials [29], photovoltaic
application [30], dermal drug delivery [31], paren-
teral, ocular, pulmonary drug delivery [32] etc.
Moreover, many researchers have also reported
targeted nanocrystals as an advancement to simple
nanocrystals, prepared by attachment/adsorption of
site-specific ligands to provide selective delivery
of payload. Hyaluronic acid-lapatinib nanocrystals
[33], transferrin-paclitaxel nanocrystals [34], al-
bumin-paclitaxel nanocrystals [35], Herceptin-
paclitaxel nanocrystals [36], triphenylphosphoni-
um-paclitaxel nanocrystals [37], iRGD-paclitaxel
nanocrystals [38] are few reported targeted nano-
crystals for anticancer activity resulted in specific
delivery to cancerous cells, increased cellular up-
take, increased cellular cytotoxicity, increased cel-
lular distribution, decreased tumor burden, in-
crease survival period, decreased dose rand dose-
related toxicity. Hence targeted/ surface-modified/
surface grafted emerged as an advancement to ex-
isting simple nanocrystals and showed better re-
sults.
In our previous review M. Kumar et al., 2019,
we explained nanocrystals role in solubility en-
hancement, various methods for preparation, chal-
lenges and advantages associated with nanocrys-
tals [39]. This review systematically summarizes
the crucial parameters which can affect the quality
of formulations and associated physicochemical
properties. In this review, we have addressed vari-
ous advantages of nanocrystals, different factors
required to be considered while preparation of
nanocrystals, and various delivery issues to be
Authors Review on Drug Nanocrystals
kept in mind. Furthermore, we have discussed
nanocrystals under different generations based on
their advancement over the last few decades. The-
se include nanocrystals without any carrier system,
nanocrystals with a carrier system, and nanocrys-
tals with surface modifiers termed as 1st genera-
tion, 2nd generation, and 3rd generation nanocrys-
tals respectively. Finally, this article summarized
various methods that can be used for the prepara-
tion and evaluation of 3rd generation nanocrystals.
2. ADVANTAGES OF NANOCRYSTALS
• Higher saturation solubility, increased adhe-
siveness, improved permeability, passive target-
ing, Carrier free colloids with 100% theoretical
drug load, long term stability, and low toxicity
risk [20, 21, 40, 41].
• Low-cost production, easy scale-up, renewable,
biodegradable [42, 43].
• Decreased variability in fed/fasted state, mini-
mum inter-subject variability, rapid onset of ac-
tion, and improved bioavailability [42].
• Better uptake via enhanced permeability and
retention (EPR) effect and none requirement to
any solubilizing agent to enhance the solubility
[34, 44].
• Passive accumulation of smaller nanocrystals of
size 50-200nm by the EPR effect [43].
• Smaller nanocrystals with neutral or hydro-
philic surface resulted in desired prolonged
blood circulation [43]. Nanocrystals of size
around 400nm tend to escape from clearance
and thus have more prolonged blood circulation
in comparison to larger sized crystals [18].
• 100nm sized nanocrystals overcome the blood-
brain barrier and enter endothelial cells via en-
docytosis, 250nm show long residence [45].
• 300-700nm form follicular reservoir [46].
• Drug targeting to specific organs and sustaining
the release [47].
3. FACTORS AFFECTING NANOCRYSTALS
SIZE
Various factors which can affect the size of sur-
face-modified nanocrystals are listed in Table 1,
which include as follows:
• Drug Concentration in Solvent System: Nano-
crystals size varies based on the concentration
Current Nanomedicine, 2020, Vol. 10, No. 1 3
of drugs in the solvent system. Initially, a de-
crease in size can be observed up to the optimal
value beyond which larger sized crystals are
formed, which is due to decreased diffusion
rate. Lu Y. et al., 2014 observed an increase in
size with the concentration of PTX (>3mg/ml)
was observed due to higher supersaturation lev-
el and decreased diffusion rate between solvent
and antisolvent. Here size increased as 3mg/ml
(268nm) < 1mg/ml (296nm) < 5mg/ml
(1670nm) < 9mg/ml (4421nm) < 12mg/ml
(4358nm) [34]. In a separate study, Chen C. et
al., 2016 revealed a decrease in size on increas-
ing concentration (2.5-5mg/ml) of the drug up
to optimal concentration (5mg/ml) beyond
which the increase in size observed (5-
20mg/ml) [45].
• Stabilizer Concentration: Stabilizer plays an
important role in the preparation of nanosized
crystals. The optimum size can be obtained
with stabilizer at their critical micelle concen-
tration, deviating from which size increment is
observed based on stabilizing property of pol-
ymer or stabilizer. An increase in size due to
stabilizer concentration above CMC is due to
the formation of multiple layers of stabilizer
around the hydrophobic surface of the drug,
while stabilizer concentration lower than opti-
mal concentration would be not enough to form
a complete layer around the hydrophobic sur-
face. So, deviation from the optimum concen-
tration of stabilizer results in the agglomeration
of drug crystals. Liu T. et al., 2018 observed a
decrease in size from 24000nm to 300nm with
an increase in the concentration of F68 [48].
Colombo M. et al., 2017 reported an increase
in size from 188 to 206nm with the increasing
concentration of Vit.E TPGS from 0.02 to
2%w/v [49].
• The Volume Ratio of Antisolvent to Solvent:
An appropriate antisolvent to solvent ratio must
be maintained to control the size of the nano-
crystals. In a study reflecting an increase in size
with antisolvent to solvent volume ratio (10:1
to 10:5), Chen C. et al., 2016 suggested that
lower nucleation rate and particle growth due to
decreased supersaturation level as a result of in-
creased solubility in the antisolvent phase was
the reason behind increase in crystal size [45].
In another study, the volume ratio for water:
ethanol (20:1) was found to be optimal. Smaller
4 Current Nanomedicine, 2020, Vol. 10, No. 1
ratio (<20:1) resulted in an increase in size due
to a higher nucleation rate and higher solvent
phase ratio. An increase in the degree of super-
saturation was also observed with the increase
in volume ratio responsible for greater nuclea-
tion rate and smaller size distribution (15,34).
Size increased as 20:1 (287nm) < 40:1 (291nm)
< 10:1 (518nm) <5:1 < (746nm).
• Type of solvent system: Solvent choice can be a
deciding factor to control the size of the nano-
crystals. The solvent having the highest solubil-
izing property and rapid diffusing property to
antisolvent for the drug could be beneficial for
obtaining nanosized crystals. Lu Y. et al., 2014
used ethanol, methanol, DMSO, methylene
chloride and ethyl acetate where increment in
crystal size was in order; methanol (280nm) <
ethanol (287nm) < methylene chloride (338nm)
< DMSO (353nm) <ethyl acetate (418nm) [34].
Mean size can increase in the sequence; metha-
nol > ethanol > propanol, where rapid diffusion
of organic phase to dispersion medium results
in the formation of nanocrystals [50].
• Stirring Speed: Speed of stirrer could affect the
size as higher speed would intensify the process
of mixing responsible for high mass transfer
and diffusion rate between different multiphase
to form a homogeneous supersaturated system
and thus quicker attainment of homogeneous
nucleation [51]. Lower speed can result in larg-
er particles due to insufficient dispersion of or-
ganic phase [52]. In comparison, higher rate
imparts greater force (additional mechanical
energy) on individual particles, which splinter
the repulsive forces between particles, as ex-
plained by Ghosh I. et al., 2012 leading to the
formation of particle aggregates [53]. Liu T. et
al., 2018 also showed a decrease in size with
elevation in stirring speed [48].
• Instrument Operation Time for size reduction:
Nanocrystal size can decrease with an increase
in treatment time for size reduction, where after
a certain period, an increase in size is observed
due to possible increase in collision rate.
Soisuwan S. et al., 2018 reported a decrease in
particle size with milling time, which becomes
constant after a certain period of time [54]. Liu
T. et al., 2018 also demonstrated the effect of
milling time where an initial decrease in size
was observed with increasing milling time up to
120min. Further increase in milling time pro-
Kumar et al.
vides a larger surface area for collision and ag-
gregation, which results in the formation of
larger crystals or aggregates. A decrease in size
was observed in order; 30min (329nm) < 60min
(300nm) < 120min (259nm) >180min (299nm)
>240min (4180nm) > 300min (3092) [48].
• Temperature Control: Cooling promotes crys-
tal formation, simultaneously preventing the
crystal growth by decreasing the dissolution of
smaller crystals and thus provides quicker stabi-
lization of nanosystem [55, 56]. In a study, it
was observed that the rapid addition of organic
phase to aqueous phase maintained at 2℃ could
result in immediate precipitation from antisol-
vent [57].
• Surface Modifier Concentration: Surface mod-
ifier plays a vital role in controlling the drug re-
lease, providing long term stability and targeted
delivery. Agrawal S. et al., 2017 experimented
to determine the optimal concentration of hya-
luronic acid as a surface modifier for the prepa-
ration of negatively charged lapatinib nanocrys-
tals. The maximum charge reversal was ob-
served at 1:4 (HA: LPT), providing desired
negative charge beyond which only increase in
size was observed [33]. Lu Y. et al., 2014
showed that an increase in viscosity due to
higher serum concentration decreased the extent
of collision and hence caused reduction in crys-
tal growth [34]. Noh J. K. et al., 2016 found
0.5mg/ml of Herceptin, optimum for maximum
adsorption to paclitaxel nanocrystals surface
[36].
• Cryoprotectant: A cryoprotectant is used to
prevent agglomeration during the process of ly-
ophilization of nanosuspension and resuspen-
sion of lyophilized nanocrystals. 5% w/v malt-
ose or mannitol [58] and 5%w/v trehalose can
be used as cryoprotectant [59]. Agrawal S. et
al., 2017 used lactose, mannitol, trehalose, su-
crose, and leucine to prevent agglomeration
while freeze-drying where 10 and 20% leucine
was found to be optimal with no lump for-
mation and are re-dispersible while other re-
sulted in an increase in size and PDI [33]. High
hydrophobic drugs form aggregates hard to re-
disperse, thus required larger but optimal sup-
porting agents like (50-100% w/v of dug) sugar,
sugar alcohols, and polymers while solidifica-
tion as the higher amount can result in lower
drug load [17].
Authors Review on Drug Nanocrystals Current Nanomedicine, 2020, Vol. 10, No. 1 5

Table 1. Overview of Optimal value observed for various Nanocrystals Related Factors.

Factor Description Reference

HPH at 20cycles 1000bar [60]

HPH at 2cycle 500bar, 1cycle 1000bar and 20 cycle 1500 bar. [61]
HPH 24 cycles 1750 bars [33]
HPH at 15cycle 20,000bar [22]
Nanoprecipitation method [40]
Probe sonication for 1min [44]
Method Probe sonication for 5 min [40]
Probe sonication for 5min [41]
Probe sonication for 10min [36]
Probe sonication for 15min, 30min, 45min [62]
Bead milling [54]
Wet Milling [21]
3mg/ml in ethanol [63]
4mg/ml in ethanol [35,44]
5mg/ml
[36,40,41,64]
5mg/ml in ethanol
6.25mg/ml ethanol [47]
10mg/ml in DMSO [65]
Drug Concentration
20mg/ml acetone [22]
100mg/ml in acetone [60]
100mg/ml [21]
120mg in DMSO [66]
700mg in 1.5ml DMSO [58]
100-500rpm [65]
200rpm [62]
300rpm [40]
300rpm [41]
500-1000rpm [22]
700rpm [21]
Speed
800rpm [34]
1000rpm [58]
1200rpm [51]
1200rpm [36]
2000-3000rpm [54]
(Table 1) contd….
6 Current Nanomedicine, 2020, Vol. 10, No. 1 Kumar et al.

Factor Description Reference

3℃ [58]
4℃ [22,34]
5℃ [42]
Temp
10℃ [60]
Room temp. [65]
40℃ [62]
Formic acid [51]
Ethanol [61]
Solvent
DMSO [65]
Ethanol, methanol, DMSO, methylene chloride, ethyl acetate [34]
Stabilizer PVP K30 [51]
Lapatinib: pluronic (1:1) [33]
Albendazole: P188 (1:1) [42]
Drug: Stabilizer Curcumin: SLS/ polaxamer (1:1) [64]
Resvaratrolo: HPMC (1:2) [61]
Paclitaxel:Pleuronic F127 (1:4) [35]
Curcumin: SLS/ polaxamer (1:1) [64]
Paclitaxel sol: water (1:5) [36,47]
Paclitaxel sol: water (1:20) [35,63]
Solvent: antisolvent
Curcumin sol: water (1:20) [22]
Nitrendipine sol: PVA sol (1:50) [60]
Nimodipine sol: water (15:1000) [58]

Fig. (1). Nanocrystals Classified as Different Generations.

4. GENERATIONS OF NANOCRYSTALS major categories (Fig. 1); nanocrystals without any

Based on the evolution of nanocrystals from carrier (1st generation), nanocrystals with a carrier
conventional nano-sized drug crystals to the modi- (2nd generation) and nanocrystals with surface
fied targeted system, it can be classified into three modification (3rd generation). These generations of
Authors Review on Drug Nanocrystals
nanocrystals have provided solutions to various
issues associated with poorly soluble drugs and
pure drug nanocrystals in order to provide systems
with distinctive advantages over its predecessors
and other conventional drug delivery platforms, as
discussed under various generations with exam-
ples.
4.1. Current Issues Associated with Poorly Sol-
uble Drug
• Poorly-aqueous soluble drug faces various limi-
tations which include the retarded onset of ac-
tion, variation in fed/fasted state, irregular dose
proportion, patient/ interpatient variability, un-
predictable absorption, lower skin penetration,
low bioavailability and the requirement of larg-
er intravascular volume for effective results [20,
67].
4.2. Challenges Associated with Conventional
Nanocrystals
• Solvent choice and its removal from formulated
nano-system, size control, and stability are im-
portant issues [34].
• High energy requirement, type of stabilizer, and
its concentration selection, lack of controlled
release and agglomeration are nanocrystal relat-
ed issues [68].
• Due to fast dissolution and lack of controlled
release kinetics, nanocrystals are reported as a
non-suitable dosage form for delivery of drugs
like anticancer drugs and thus require modifica-
tion [69].
• Nanocrystals if not coated to cover hydrophobic
surfaces can exhibit rapid clearance from blood
circulation via phagocytosis by the mononucle-
ar phagocytic system (MPS) [22, 63].
• Administered nanosuspension is prone to up-
take by liver, kidney, lung, spleen, and translo-
cate to various tissues [22].
• Possible nano-toxicity could be due to cellular
uptake via endocytosis [20].
4.3. First Generation
This category involved the preparation of drugs
as nanocrystals, which can be an aid to increase
aqueous solubility, improve dissolution velocity
and enhance the rate of permeation, and hence im-
proving activity and efficacy, thus resulted in in-
Current Nanomedicine, 2020, Vol. 10, No. 1 7
creased bioavailability of the drug. Here, the drug
is first nanosized by means of high energy input
resulting in a thermodynamically unstable system
and simultaneously stabilized by the use of steric
(polymeric) or electrostatic (surfactants) stabilizers
to give a stable system.
4.3.1. First-Generation Nanocrystals: Principle
and Advantages
Pure drug nanocrystals are likely to undergo
fast dissolution due to their nano-size. However,
they are stabilized with the help of polymer or sur-
factant or combination of both, and hence, do not
dissolve instantly upon administration instead dis-
play sustained and controlled release possibly due
to incorporated stabilizers [71]. Nanocrystals act
distinctively on systemic administration, primarily
bypassing various barriers where dissolved drugs
follow passive diffusion while remaining nano-
crystals undergo absorption through clathrin or
caveolin mediated endocytosis. Besides, nanocrys-
tals in blood circulation experiencing phagocytotic
uptake will disintegrate, dissolve, and release the
drug, which will come back to circulation due to
concentration gradient and follow passive diffu-
sion [72].
In the systemic circulation, pure drug nanocrys-
tals without any stabilizers were prone to binding
with opsonins, which induces the process of phag-
ocytosis by forming a bridge between the hydro-
phobic surface of nanocrystals and phagocytic
cells, followed by removal through MPS [73].
Thus, to increase blood circulation, decrease the
prior release of drugs from nanocrystals and pre-
vent clearance via MPS, a protective coating by
the use of PEGylated stabilizers is done to cover
the hydrophobic surface. This hydrophilic layer
also provides increased circulation time by repel-
ling opsonins and hence delayed recognition by
MPS. Furthermore, the clearance of these
PEGylated nanocrystals by organs like liver, kid-
ney, spleen, etc is decreased so that the amount of
administered drug is in the circulation for maxi-
mum duration of time [73]. For example, pure cur-
cumin nanocrystals administered by i.v. injections
get cleared from the blood and accumulated in the
lung, spleen, kidney, and mainly liver due to RES
uptake, leaving only about 33% NC in blood circu-
lation at 4h [22]. A study was also conducted by
Hollis C.P. et al., 2013 to see the biodistribution of
tritium labeled pure PTX NC without any stabi-
lizer and integrated with dye. A high concentration
8 Current Nanomedicine, 2020, Vol. 10, No. 1 Kumar et al.

Table 2. Simple Nanocrystals with Multiple Advantages.

Nanocrystals Use Advantage Reference

Superior activity, 9 fold aqueous solubility, and

superior anthelmintic activity with lower paral-
[51]
Albendazole Anthelmintic (1982) ysis and death time (49 and 71 min) as com-
pared to the coarse drug (75 and 104 min).
Enhanced chemo-prophylactic activity [42]
4.73 times bioavailability, good re-
Allisartan Isoproxil - [17]
dispersibility, high drug load
Increased permeability, cellular uptake, and
Clarithromycin Anticancer [54]
caco-2 cell motility
Meloxicam - 44 fold AUC0-24, increased dermal delivery [70]
Nimodipine - 2-6 fold bioavailability than Nimotop [58]
Nisoldipine - Superior absorption [59]
Anticancer Drug (US FDA
Paclitaxel Improved activity [62]
1992)
neurodegenerative disease
20-S Proto Panaxadiol Improved brain delivery [45]
and intracranial tumor
1.37 fold solubility, 2 to 2.75 times permeation
Pramipexole - [46]
with 34% follicular based permeation

of PTX NC was observed in organs like liver,
spleen, lungs suggested for possible clearance of
nanocrystals. However, the liver may act as a de-
pot, releasing drugs back into circulation for redis-
tribution. The observed entry of nanocrystals to
tumors can be via enhanced permeation and reten-
tion effect, possibly due to available large inter
endothelial pores and disorganized growth of tu-
mors. However, still, the poor diffusion was ob-
served [74]. In another study, surface modification
of PTX NC with DSPE PEG 2000 by Wang D. et
al., 2019 revealed an improved systemic circula-
tion and slower drug release. DSPE-PEG 2000-
PTX-NC helped to achieve about 2 fold AUC and
at the same time, reducing about 2 fold clearance
compared to simple PTX-NC [75]. In a separate
study done by Liu Y. et al. 2017, baicalin nano-
crystals consisting of tween 80 and TPGS as a sta-
bilizer was prepared. These stabilizers provided a
hydrophilic protective layer around nanocrystals,
thus minimizing opsonization. Moreover, findings
revealed that apolipoproteins (Apo E 67 and Apo J
7.9%), fibrinogen (0.1%) and Igy (6.4%) were ad-
sorbed on the surface of prepared nanocrystals.
Adsorption of apolipoproteins on the surface of
baicalin nanocrystals further decreased the opsoni-
zation, minimized hepatic uptake, increased blood
circulation, improved uptake and penetration to the
brain via LDL receptor-mediated endocytosis.
About 6.67 and 2.59 times, drug concentration was
observed in the brain from Tween80/TPGS-NC
compared to coarse drug and Tween 80-NC re-
spectively [73].
4.4. Second Generation
Second generation nanocrystals based delivery
system focusses on the controlled release of drugs
at a specific site or tissue such as lung, nasal and
skin, etc. Table 3. Here the prepared nanocrystals
are incorporated inside a carrier system to provide
targeted delivery with desired site specific release.
4.4.1. Advantages
• 2nd generation nanocrystals include nanocrys-
tals loaded drug delivery systems to give a
modified system with the desired action like the
controlled release, sustained release, targeted
delivery, etc. Liu G. et al., 2016 loaded zein
microspheres with 10-hydroxy camptothecin
Authors Review on Drug Nanocrystals
Table 3.
Nanocrystals Delivery System
In-Situ Hydrogels for
Azelic acid
topical delivery
Luliconazole Hydrogel for topical delivery
Hydrocamptothecin Zein based nano complex
Spray-dried Liposome for
Ciprofloxacin
inhalation
Budenoside Microparticle for inhalation
Chitosan-based Microparticles for
Cinaciguat
pulmonary delivery
nanocrystals to give a targeted controlled and
sustained release system that protected the
nanocrystals from degradation at the same time
preventing from any possible toxic effects [69].
Li T. et al., 2018 used PVA hydrogel-based
microspheres to incorporate fenofibrate nano-
crystals, which outcome to a synergistic effect
of sustained-release with lower fluctuation in
plasma concentration and thus provided higher
bioavailability compared to free drug or
Lipanthyl [68].
• These nanocrystals loaded carrier system can
also be used for targeted delivery. Yang H. M.
et al., 2011 encapsulated doxorubicin and iron
oxide nanocrystals into multifunctional poly
aspartic acid-based nanoparticles which result-
ed in specific delivery to cancerous cells and
better resolution for MRI [80].
• 2nd generation nanocrystals may provide an ef-
ficient delivery system for local action also. Liu
T. et al., 2018 loaded budesonide nanocrystals
into hyaluronic acid-based microparticles. The
resultant delivery system prolonged the reten-
tion on the trachea and combated mucociliary
clearance via mucoadhesive property [48]. Ni
R. et al., 2017 formulated a sustained pulmo-
Current Nanomedicine, 2020, Vol. 10, No. 1 9
Observation Reference
Delivery to deeper skin layers, A similar response
in half of the dose to commercially available [76]
cream.
Increased antifungal activity, deepen penetration
[77]
and increased accumulation to various skin layers
predictable and controlled release high drug load,
overcame instability and low diffusion, translo- [66]
cated close or inside to nucleus
Stability maintained following rehydration, supe-
rior aerosol performance, prolonged-release, ex-
[78]
ceeded MIC by 10 factors for the treatment of
biofilm and lung infection
Prolonged retention on the surface of the tracheal
tube, provides a slow release which is favorable [48]
for local therapy
Provided sustained delivery, promising aerosoli-
[79]
zation, high drug load
nary drug delivery system with cinauguat nano-
crystals in chitosan-based microparticles [79]
and Wu H. et al., 2018 decorated ceria nano-
crystals on mesoporous silica nanoparticles.
These 2nd generation NCs exhibited recoverable
ROS scavenging activity to protect against oxi-
dative stress and catalytic mimetic activity
greater than free nanocrystals. Immobilization
of ceria nanocrystals on silica nanoparticles en-
hanced the quality of skin healed, limited the
scar formation, decreased cellular inflamma-
tion, senescence and accelerated the wound
healing by the nano-bridging effect which
closed the deep wound rapidly with negligible
cytotoxicity [81]. Feng L. et al. 2018 also en-
capsulated iridium nanocrystals into PEG-
modified liposomes. The resulted delivery sys-
tem protected the iridium nanocrystals having
catalase activity for H2O2 from molecules con-
taining thiol group, provided long circulation
half-life, efficient accumulation in tumor cells
via EPR effect and relief from tumor hypoxia
while conducting radiotherapy and thus en-
hanced the efficacy [82].
• Nanocrystals loaded carrier systems have sev-
eral distinct advantages over other carrier sys-
tems, providing higher drug load and controlled
10 Current Nanomedicine, 2020, Vol. 10, No. 1
release. Silica lipid hybrid based microparticles
loaded with ziprasidone nanocrystals prepared
by Meola T. R. et al., 2018 improved the drug
loading capacity (17 fold), solubilization (4.7
fold) and delivery [83]. Similarly, Wang H. et
al., 2017 controlled the release, increased the
drug loading (from 2.4 to 15.3%) and improved
the encapsulation (from 48.5 to 91.9%) of
breviscapine and breviscapine NCs by prepar-
ing PLGA microparticles containing breviscap-
ine nanocrystals [84].
4.5. Third Generation
Nanocrystals have evolved to a great extent
over the time. Many researchers have utilized var-
ious types of ligand, such as Herceptin, Hyaluronic
acid, peptides (TAT, iRGD, PTD, MPG), folate,
D-glucosamine, and aptamers to modify the sur-
face of nanocrystals and provide selective delivery
to a specific site. Ligands recognize specific recep-
tors overexpressed on targeted cells, minimize dis-
tribution to no-specific cells or organs and thus
decrease possible toxic effects [85].
Surface modifications in 3rd generation nano-
crystals is done by adsorption or chemical means.
Chemical change on the surface of cellulose nano-
crystals by oxidation to introduce carboxyl group
responsible for hydrogen bond formation with rep-
aglinide or adsorption of chitosan to nanocrystals
surface by inotropic gelation retarded the drug re-
lease giving a sustained and controlled release sys-
tem, modified cellulose nanocrystals (CNC) sur-
face with purpurin-18, and PEI-chlorin 6 attached
CNC by means of covalent bond between oxidized
glucose unit on the surface and the amine group of
PEI, are few instances of modified NCs [43, 67].
Similarly, Choi H. W. et al., 2006 used the vinyl
phosphonic acid group (VPA) to alter the surface
of hydroxyapatite nanocrystals (HAp), which en-
hanced the colloidal stability, dispersion proper-
ties, and bio-adhesion [86]. Han X et al., 2018
used Triphenylphosphonium cation-conjugated
Brij 98 to modify paclitaxel nanocrystals (PTX-
NC), which elevated drug accumulation in mito-
chondria, improved penetration, and targeted de-
livery to MCF-7/ADR cells, thus providing effec-
tive growth inhibition to drug-resistant cancer
[37].
Surface modification can also be done by elec-
trostatic interaction with surface charge to obtain
targeted nanocrystals. The use of positively
Kumar et al.
charged Poly (allylamine-hydrochloride), PAH
and negatively charged Poly (sodium-4-styrene-
sulfonate), PSS by Choi J. S. and Park J. S.,
2016 to modify the surface of PTX-NC improved
interaction with bovine serum albumin, which en-
hanced the cell cytotoxicity by improved cellular
uptake. PAH-PTX-NC had 8 fold increase in in-
teraction with albumin than PTX-NC, as a result,
cell internalization increased up to 2.5 fold of PSS-
PTX-NC and 1.5 fold PTX-NC [40].
4.5.1. Advantages
• 3rd generation nanocrystals provide targeted de-
livery to the desired area without any prior dis-
semination and drug loss while transportation
(Table 4). Noh J. K. et al., 2016 grafted
paclitaxel nanocrystals with Herceptin acting as
a targeting ligand that recognizes HER2 recep-
tors in breast cancer cells, which resulted in
prolonged blood circulation, avoided drug loss
before reaching targeted organ and enhanced
cellular uptake with better cell growth inhibi-
tion [36].
• They ensure maximum concentration at a spe-
cific area, targeted for the desired action. In an
experiment conducted by Park J. et al., 2016,
albumin was adsorbed on paclitaxel nanocrys-
tals surface, which reduced uptake by J774A1
macrophages and increased the uptake to
B16F10 melanoma cells. An increase in PTX
concentration in tumor cells was observed by
modified nanocrystals treated cells (27.4µg/ml)
than unmodified PTX (13.8µg/ml) [35].
• Surface modification of nanocrystals also im-
proved the activity and potency. De Castro,
Daniele Oliveira, et al. 2015 prepared rosin
grafted cellulose nanocrystals, which exhibited
stronger antibacterial activity [87]. Ferreira F.
V. et al., 2018 used adipic acid to modify the
surface of cellulose nanocrystals, which im-
proved potency by facilitating the interaction
with hydrophobic surfaces [88]. Lu Y. et al.,
2014 modified the paclitaxel nanocrystals
(PTX) surface with serum protein transferrin
(Tf), where modified PTX-Tf resulted in higher
tumor inhibition (45%) than unmodified PTX-
NC (28%) [34].
• These targeted nanocrystals provide selective
delivery to the desired area via some specific
receptors. In the past few years, many targeted
nanocrystals have been reported for targeting
Authors Review on Drug Nanocrystals
tumors or cancers of various types. Agrawal S.
et al., 2017 prepared hyaluronic acid (HA)
coated lapatinib nanocrystals (HA-LPT-NC)
involved in active targeting via CD44 receptor,
overexpressed in cancer cells. HA provided a
negative charge which improved pharmacoki-
netics parameters, prolonged circulation half-
life by providing hydrophilic surface, and thus
protected from rapid clearance by preventing
opsonization. HA-LPT NC found to have 52%
and 83.32% reduction than LPT-NC and free
LPT [33]. Choi J. S. and Park J. S., 2017
modified docetaxel nanocrystals (HCT-DTC-
NC) with Herceptin, which improved drug re-
lease, enhanced cellular uptake, and cytotoxici-
ty in MCF-7 cells [44]. Sohn J. S. et al., 2017
modified paclitaxel nanocrystals with Apo-
transferrin (Tf) and hyaluronic acid (HA) to
provide targeted delivery and better cell growth
inhibition. Tf-PTX-NC undergoes Tf receptor-
mediated endocytosis, overexpressed in cancer
cells requiring high iron demand. Whereas HA-
PTX-NC binds to CD44 (overexpressed in
breast and gastric cancer), RHA MM (receptor
for HA mediated motility) and ICAM-1 (inter-
cellular adhesion molecule) for tumor targeting.
HA-PTX-NC observed to have higher cellular
uptake than Tf-PTX-NC and PTX-NC [41].
Zhan H. et al., 2017 grafted XQ1 tumor-
targeting peptide onto the PDA coated camp-
tothecin nanocrystals (CPT-NC) surface to
form modified nanocrystals, which binds spe-
cifically to transferrin receptors and thus in-
creased the anticancer activity on A549 (>125)
and Hela cancer cells (2.2) in comparison to
unmodified CPT-NC [65].
• Brain targeted nanocrystals have also been re-
ported, which targets the brain or brain tumors
via adsorptive mediated transcytosis or recep-
tor-mediated endocytosis by means of various
receptors overexpressed on BBB and surface of
tumors present in the brain. PTX NC was modi-
fied with RGD to obtain superior intra-tumor
internalization via receptor-mediated endocyto-
sis. RGD-PTX-NC showed 2.3 and 2.2 fold in-
crease in drug accumulation than uncoated NC
and PEGylated-NC, respectively [38].
5. METHODS OF PREPARATION
Modified 3rd generation nanocrystals can be
prepared by modification on the surface of pre-
Current Nanomedicine, 2020, Vol. 10, No. 1 11
pared nanocrystals to improve the efficacy of the
drug delivery system.
5.1. Preparation of Nanocrystals
The preparation of nano-crystalline drugs is
mainly based on two approaches i.e. top-down and
bottom-up. The top-down approach involves na-
nosization via high energy forces provided by var-
ious mechanical instruments like media mill, high-
pressure homogenizer, and probe sonication. In
contrast, the bottom-up approach is preparation or
synthesis of nano-sized particles by the process of
nucleation, triggered with removal of solvent or
use of an antisolvent [20]. Simple nanocrystals can
be prepared by various methods, as discussed in
our previous review, M. Kumar et al., 2018 [39],
later surface modified to obtain grafted nanocrys-
tals.
5.2 Surface Modification
It is the process of refinement and modulation
done to the surface to improve the physicochemi-
cal property, stability and targeting efficacy of the
drug. It can be done mainly by two approaches,
namely; adsorption method and chemical modifi-
cation.
5.3. Adsorption Method
Prepared nanocrystals were re-dispersed in sur-
face modifier solution for an interval of time while
stirring to obtain surface-modified nanocrystals
[40]. Choi J. S. and Park J. S., 2016 conducted
adsorption of poly-allylamine hydrochloride and
poly-sodium-4-styrene sulfonate to the surface of
PTX-NCs [40]. Noh J. K. et al., 2016 adsorbed
Herceptin to paclitaxel nanocrystals by adsorption
method [36]. Rydberg H. A. et al., 2016 conduct-
ed an adsorption study with DSPE-PEG on felodi-
pine and griseofulvin nanocrystals [21].
5.4. Chemical Modification
Surface grafting is done based on functional
groups available on the surface of nanocrystals
[87]. Etherification, oxidation, amidation, esterifi-
cation and graft polymerization are few of the fre-
quently used chemical modification methods [29].
Ntoutoume G. MA N. et al., 2017 used cellulose
nanocrystals surface to graft with PEI where glu-
cose units on cellulose surface were initially oxi-
dized, which are later used to bound PEI by reduc-
tive amination to form CNC-PEI suitable for
12 Current Nanomedicine, 2020, Vol. 10, No. 1 Kumar et al.

Table 4. Surface Modified Nanocrystals and their Advantages.

Nanocrystals Use Surface Modifier Advantage Reference

Anatase V 2O 5 Modified Adsorption [89]

Dopamine peptide graft- Rapid cross membrane translocation via
Camptothecin Anticancer [65]
ed polydopamine endocytosis, tumor-targeted delivery
Better interaction with hydrophobic
Adipic acid [88]
matrices
Polyaminated chlorin 6 Improved cancer cell targeting [43]
Cellulose
Rosin Strong antibacterial activity [87]
Thiol containing castor
Improved delivery [90]
oil
Cellulose loaded Specific gene si- Protected siRNAs from degradation,
PEI [91]
with siRNAs lencing sequence favored delivery,
Cellulose NC load- Oral hypoglycemic
Chitosan Sustained and controlled release [67]
ed with Repaglinide agent
Cellulose NC load- Improved Antiproliferative effect on
Anticancer Beta cyclodextrin [92]
ed with curcumin colorectal and prostatic cancer cells
Cuprous telluride Enhanced cellular uptake, synergistic
NC loaded Doxoru- Anticancer DSPE PEG 2000 kill effect, good capacity to load hydro- [93]
bicin phobic drug, prolonged circulation
Anticancer, anti-
Curcumin inflammatory, anti- Polaxamer 188 Increased circulation time [22]
oxidant
Anticancer Drug Good Cellular Uptake and Cytotoxicity
Docetaxel (DTX) Herceptin (HCT) [44]
(US FDA 2006) for HER2 bearing cells
Felodipine and DSPE PEG 2000 and Prolonged circulation time and targeted
[21]
Griseofulvin DSPE PEG 5000 delivery
Substitute for De- Polyvinyl phosphonic Enhanced stability, Excellent Dispersion
Hydroxyapatite [86]
fective Bones acid Properties
Selective cellular internalization, mini-
Hydroxy Camptoth- Folate conjugated poly- mum drug release in extracellular cells
Anticancer [66]
ecin dopamine or during circulation, Superior tumor
suppression
Improved pharmacokinetic parameters,
Lapatinib Anticancer Hyaluronic acid targeted delivery, superior anticancer [33]
activity
Decreased MPS uptake, prolonged cir-
Mn-Zn Ferrite DSPE PEG 2000 culation, passive targeting via EPR [94]
effect
Nitrendipine Chitosan Increased stability and bioavailability [60]
(Table 4) contd….
Authors Review on Drug Nanocrystals Current Nanomedicine, 2020, Vol. 10, No. 1 13

Nanocrystals Use Surface Modifier Advantage Reference

Poly(allylamine-
hydrochloride), Higher Cell Uptake and
[40]
Poly(sodium-4-styrene- Cell Cytotoxicity
sulfonate)
Apo-transferrin, hyalu- Higher cell permeability and cellular
[41]
ronic acid uptake, better cell growth inhibition
Decrease uptake by MPS increased
Albumin [35]
uptake to cancer cells
Decreased prior burst-release, improved
DEPE MPEG [47]
cellular uptake, and cell killing ability
Anticancer Drug
Paclitaxel Enhanced antitumor activity, targeted
(US FDA 1992) Herceptin [36]
delivery
Lowered the MPS uptake, enhanced
blood circulation, 3-4 times accumula-
tion,
Pluronic F68 Provided a hydrophilic steric barrier to [63]
paclitaxel nanocrystals which shielded it
from recognition, opsonization and thus
elimination by the immune system.
About twice tumor inhibition rate, pro-
Serum protein transferrin [34]
longed circulation decreased prior loss
Titanium Oxide PEG Improved photocatalytic activity [95]

loading siRNAs associated by ionic interaction
[91]. Ntoutoume G. MA N. et al., 2015 used cati-
onic beta-cyclodextrin (CD) prepared by reacting
cyclodextrin with glycidyl-trimethyl-ammonium
chloride for grafting to the surface of anionic cel-
lulose nanocrystals (CNC) via ionic interaction
[92]. Choi H. W. et al., 2006 modified hydroxy-
apatite surface having abundant P-OH as a func-
tional group by grafting with vinyl phosphonic ac-
id [86].
6. EVALUATION
Modified nanocrystals can be evaluated mainly
for residual solvent level (if involve use of organic
solvent), particle size and zeta potential, surface
modifier concentration, specific surface area, sta-
bility, drug release, permeation study, etc. (Table
5).
6.1. Quantification of Residual Solvent
Quantification for residual solvent should be
determined to check whether the solvents are with-
in the acceptable limits, so as to ensure the safety
of the system. The residual level of the solvent
used in the preparation of nanocrystals can be
quantified by gas chromatography. Koradia D. K.
et al., 2018 found 481ppm formic acid in the pre-
pared nanocrystals, well below the acceptable lim-
its (<5000ppm), ensuring the safety of NCs [51].
6.2. Determination of Particle Size and Zeta Po-
tential
Increase in particle size and change in zeta
potential can be observed due to surface modifi-
cation of drug nanocrystals. Thus, the determina-
tion of particle size and zeta potential with the
help of zeta-sizer is used to confirm the surface
modification (Table 6). Maximum charge rever-
sal was observed for hyaluronic acid coating (at
1:4 HA: LPT) to lapatinib nanocrystals required
for complete coating which provided hydrophilic
layer, negative potential for intravascular admin-
istration and higher tumor-suppressing activity
(at 7 doses of 20mg/kg instead of 30mg/kg re-
quired with albumin-based nanoparticle for simi-
lar action) [33].
14 Current Nanomedicine, 2020, Vol. 10, No. 1 Kumar et al.

Table 5. Analytical Techniques Available for Characterization of Surface Modified Nanocrystals.

Characteristics Analytical Technique Reference

Dynamic Light Scattering (DLS), Fluorescence Correlation Spectroscopy

(FCS), Near Field Scanning Optical Microscopy (NSOM), Scanning Electron
Microscopy (SEM), Transmission Electron Microscopy (TEM), Scanning
Size and size distribu-
Tunnelling Microscopy (STM), Atomic Force Microscopy (AFM), Nuclear [96]
tion
Magnetic Resonance (NMR), X-Ray Diffraction (XRD), Gel Electrophoresis
(GE), Column Electrophoresis (CE), Raman Spectroscopy, Surface Enhanced
Raman Spectroscopy (SERS)
Zeta potential, Attenuated Total Reflection Fourier Transform Infrared (ATR-
Charge [96,97]
FTIR), GE, CE, SPM
SEM, TEM, STM, AFM, XRD, Small Angle X Ray Spectroscopy (SAXS),
Shape [96,98]
NSOM
Structure XRD, IR, NMR, DSC, AUC, Raman, MS, STM, AFM, SAXS [96,98]
X-ray diffraction (XRD), transmission electron microscopy (TEM), ultravio-
let-visible (UV–Vis) spectroscopy, photoluminescence spectroscopy (PL),
Phase and Morphology [6,7]
Fourier transform infrared (FT-IR) spectroscopy and X-ray photoelectron
spectroscopy (XPS), The energy dispersive spectrometry (EDS) analysis
Composition MS, NMR, TGA, Energy-dispersive spectroscopy (EDS) [5,96,98]
Stability Zeta potential, DSC, TGA, MS, FTIR, RS [96,97]
[Lin et al.,
Surface Area BET
2014]
Surface Coating Com-
MS, FTIR, NMR, SPM, XPS [97]
position
Surface Coating Cover-
AFM, AUC, TGA [97]
age
Surface Functionaliza- X-ray photoelectron spectroscopy (XPS), Energy-dispersive X-ray spectros-
[96,98]
tion copy (EDX), Fluorescence microscopy, SEM, TEM, FTIR, MS,
Surface Roughness Atomic force microscopy (AFM), AFM-IR [98]
Corona (Thickness) DLS, FCS, TEM [96]
Corona (Affinity) Circular Dichroism (CD), Size-exclusion Chromatography [96]
Mechanical Properties Mechanical Tester [98]

6.3. Quantification of Surface Modifier on
Nanocrystals Surface
Surface modified nanocrystals are centrifuged
at 12000rpm for 10min. The supernatant is col-
lected, and surface modifier on nanocrystals can
be determined by the indirect method of estimating
the surface modifier concentration in supernatant
and subtracting it from initial concentration [44].
Drogat N. et al., 2012 determined the quantity of
polyamine chlorin 6 used as a surface modifier on
the CNC surface using UV-absorption spectropho-
tometry, which revealed complete grafting [43].
Gao W. et al., 2016 determined F68 concentration
by use of HPLC [63].
6.4. Determination of Specific Surface Area
Specific surface area and pore size can be de-
termined by Brunauer-Emmett-Teller instrument.
The specific surface area of hydroxyapatite sur-
face-modified nanocrystals prepared by Choi H.
Authors Review on Drug Nanocrystals Current Nanomedicine, 2020, Vol. 10, No. 1 15

Table 6. Change in Particle Size and Zeta Potential with Surface Modification.

Particle size Zeta potential

Drug Reference
Before After Before After

HCT-DTX-NC DTX-NC HCT-DTX-NC

Docetaxel DTX-NC 516nm [44]
540nm -9.6mV 10.4mV

Hap-NC - VPA-Hap- NC -
Hydroxyapatite Hap-NC 80nm VPA-Hap- NC 90nm [86]
1.87mV 6.23mV
CNC-PEI 600 226NM, CNC-PEI 600
CNC 141.5nm CNC-PEI-siRNAs CNC -51mV 36.9mV, CNC-PEI- [91]
287nm siRNAs 2.55mV

Cellulose OXCNC 78-

200nm, CNC- OXCNC-CHNP 235- CNC-CHNP 30- OXCNC-CHNP
[67]
CHNP 215- 330nm 40 2326mV
338nm

Lapatinib LPT 236nm HA-LPT-NC 280nm LPT 24mV HA-LPT-NC -18mV [33]

Nitrendipine NC 175nm Chitosan-NC 180nm -6.58mV 40mV [60]

HA-PTX-NC -
PTX-NC HA-PTX-NC 533nm, PTX-NC
16.6mV, [41]
301nm Tf-PTX-NC 504nm -2.7mV
Tf-PTX-NC -15.9mV

PTX-NC - F68-PTX-NC -
PTX-NC 174nm F68-PTX-NC 187nm [63]
14.8mV 30.8mV

PTX-NC - HCT-PTX-NC
PTX-NC 239nm HCT-PTX-NC 243nm [36]
5.14mV 11.2mV
Paclitaxel
PTX-NC Alb-PTX-NC
[35]
321nm 358nm

PSS-PTX-NC -
PSS-PTX-NC 405nm, PTX-NC - 22.7mV,
PTX-NC 320nm [40]
PAH-PTX-NC 407nm 2.4mV PAH-PTX-NC
19.3mV

W. et al., 2006 was found to be 70m2/g of nano-
crystals sample [86].
6.5. Stability Study
Stability studies are performed by storing modi-
fied nanocrystals at 4 and 37℃ for a 6 months.
Samples are analyzed at fixed intervals, for parti-
cle size and zeta potential, changes in crystalline
behavior, the extent of drug leaching, change in
pH, and shelf life of nanocrystals to determine the
extent of stability. The stability study is carried out
both in solution (re-suspended) and solid-state (ly-
ophilized NCs) [44].
6.6. Fourier Transform Infrared (FT-IR) Spec-
trometry and TGA
FTIR study is done to determine the interaction
between drug, stabilizer, and surface modifier. The
presence of any new chemical/functional groups or
ligands on the surface of the nanocrystals as a re-
sult of surface modification is observed. Infrared
spectra obtained using an IR-spectrophotometer in
the range of 4000–400cm−1 and thermogravimetric
analysis (TGA) is also done to determine any new
bonds formed due to excipient drug or intra-
excipient interactions [88]. Choi H. W. et al.,
2006 observed 1030, 1080 cm-1 for unmodified
16 Current Nanomedicine, 2020, Vol. 10, No. 1
HAp due to the presence of phosphate group while
the additional band at 2850, 2950 cm-1 for modi-
fied HAp due to grafting with PVPA [86]. Sohn J.
S. et al., 2017 reported extra Peaks at 1635.7, 1643
&1643.9 cm-1 for Tf-PTX-NC, 1600.5, 1630.0
&1630.1 cm-1 for HA-PTX-NC instead of all
peaks with PTX-NC [41]. FTIR study is also be
made to confirm the complete encapsulation of
drugs inside a carrier system, as it causes the ex-
tinction of peaks for the coarse drug [77].
6.7. Differential Scanning Calorimetry (DSC)
DSC analysis performed using a thermal ana-
lyzer to validate the surface modification. DSC
instrument is operated in an inert nitrogen atmos-
phere (40ml/min) at a scan rate of 10 ◦C/min from
10 ◦C to 350 ◦C to determine any modification or
change occurred prior to or during the storage of
nanocrystals (41,60). A shift in endothermic peaks
can be observed due to changes in size. Usually, a
decrease or downward shift in peak is observed
with size reduction, like endothermic peaks ob-
served at 84.8 & 163.6℃ for PTX-NC, 83.3 &
163.3℃ for HA-PTX-NC and 43.7 & 165 for Tf-
PTX-NC [41].
6.8. Field Emission Scanning Electron Micros-
copy (FE-SEM)
Morphological evaluation of surface-modified
nanocrystals can be conducted using FE-SEM.
Surface modified nanocrystals spread onto a car-
bon tape coated with gold is viewed under an ac-
celeration voltage of 5.0 kV. Change in particle
size and shape might be observed due to surface
modification [9]. Sohn J. S. et al., 2017 found par-
ticle size at 302 nm for Tf-PTX-NC while for HA-
PTX-NC at 339 nm [41]. The dimensions of
80nm*15nm (length*width) for Hydroxyapatite
modified nanocrystals were observed using FE-
SEM [86].
6.9. Powder X-ray Diffraction (PXRD)
The samples are analyzed using an X-ray dif-
fractometer with Cu K radiation at a specific 2-
theta range to determine the crystallographic pat-
tern of prepared nanocrystals [60]. The diffraction
peaks with various theta and bravais lattice values
(h, k, l) can be analyzed [77]. Crystalline peaks
can follow a reduction in intensity due to adsorp-
tion or grafting of surface modifier on nanocrystals
surface [36].
Kumar et al.
6.10. In-Vitro Drug Release Study
It can be performed by checking the release be-
havior of nanocrystals kept inside the dialysis bag,
immersed in body simulated fluid (e.g. phosphate
buffer pH 7.4, 1.2, 6.8 and acetate buffer pH5.5) at
50rpm and 37±0.5℃ for about 24 to 48 hr. The ali-
quots are withdrawn at predetermined intervals of
time and replaced by the same volume of fresh
media. The samples are centrifuged at a high rpm
to collect supernatants, filtered, and further used
for the determination of drug content with the help
of suitable analytical methods like high-
performance thin-layer chromatography (HPLC)
[41].
7. INDUSTRIAL STATUS OF NANOCRYS-
TALS
From the early stages of development of na-
noscale drug delivery platforms, seldom, the drugs
used to reach the target tissues while mostly circu-
lated throughout the systemic circulation (>90%).
So, there was a conscious effort by researchers from
both industry and academia to develop the next
generation of the nanoscale platform, providing tar-
geted delivery. The search for nanoscale formula-
tion, stable, specific and safe enough for transla-
tional value has led us to the nanocrystals. Many
companies are engaged in the formulation of inno-
vative nanocrystalline drug delivery systems. Re-
cently, an Ireland based company, Alkerme’s “Ari-
piprazole Lauroxil NanoCrystal Dispersion” (AL-
NCD), a novel investigational product for the treat-
ment of schizophrenia has been accepted for filing
by the U.S. Food and Drug Administration, June 30,
2018, under the Prescription Drug User Fee Act.
[99]. A novel use of nanocrystal technology by Sci-
entists at the University of Nebraska Medical Center
was reported that involved preparation of dolute-
gravir nanocrystals. Prepared nanocrystals were able
to penetrate hidden HIV reservoirs with facilitated
entry into lymph nodes, bone marrow, and persisted
for months showing an extended life. The formed
delivery system released the drug only in surround-
ing tissues on entering the macrophages and was
able to withstand temperature variations inside the
body [100]. SILCRYST™ nanocrystals by Smith
and Nephew's used in Acticoat™ dressings showed a
sustained release of silver ions for up to 7 days. They
demonstrated to have broad-spectrum antimicrobial
activity inhibiting methicillin-resistant Staphylococ-
cus aureus and vancomycin-resistant enterococcus
Authors Review on Drug Nanocrystals
Fig. (2). “King Design” for various Nanocarriers.
[101]. EquivaBone introduced by Zimmer Biomet
Etex, which incorporated the use of nanocrystalline
calcium phosphate and hydroxyapatite crystals
found to be an ideal substitute for new bone growth
[102]. Ryanodex brought in market by Eagle Phar-
maceuticals as nanocrystalline suspension of dan-
trolene was able to attain required concentration
(150 times) with quick reconstitution (within 10sec)
and thus can be used efficiently for life-threatening
malignant hyperthermia [103].
8. AUTHORS INSIGHT ON THE TOPIC
Based on the evolution of nanocrystals from its
origin, to the formulation of the targeted delivery
system for a specific action, nanocrystals here
have been discussed under three generations; sim-
ple nanocrystals, nanocrystals inside a delivery
system and modified nanocrystals. Surface modi-
fied nanocrystals can be a new emerging targeted
drug delivery platform where the surface can be
changed with the help of suitable polymer, surfac-
tant, ligands and a moiety required to attain target-
ed delivery (Fig. 3). Surface modified nanocrystals
are prepared by top-down and down-up approach-
es where surface modification can be achieved via
adsorption method or chemical modification to the
surface of nanocrystals based on the presence of
functional groups. Various factors affecting the
formulation of nanocrystals which can affect the
size of prepared nanocrystals includes drug con-
centration, stabilizer concentration, solvent to anti-
solvent ratio, a drug to stabilizer ratio, stirring
speed, instrument type and its application time,
temperature, cryoprotectant and surface modifier
concentration.
Current Nanomedicine, 2020, Vol. 10, No. 1 17
The above review suggests that 3rd generation
nanocrystals is a potential universal approach for
selective drug delivery and can be superior to oth-
er nano-carrier systems as picturized in the “King
Design” (Fig. 2). King design places the targeted
nanocrystals at the top of all other methods due to
higher drug payload and improved therapeutic ac-
tions. Nanocrystals inside a delivery system are
placed below 3rd generations, which are found to
have higher observed potency as compared to the
same delivery system containing the coarse drug.
At the bottom of the “King Design” simple nano-
carrier systems are placed, above that are simple
nanocrystals due to comparatively higher activity
and stability.
3rd generation targeted nanocrystals are pre-
pared by surface modifications by ligands such as;
Peptides, Adipic acid, Rosin, DEPE PEG, Serum
proteins, Transferrin, Hyaluronic acid, Herceptin,
Aptamers, etc. (Fig. 3). Surface modification could
prove to be an effective way to prevent cellular
uptake of administered nanocrystals by liver, kid-
ney, lung, spleen and various tissues via MPS ef-
fect and overcome prior drug release and dissolu-
tion while in blood circulation (non-target site).
Modifications on the surface allow desired actions
like passive and active targeting, selective cellular
internalization, and higher permeation, improved
pharmacokinetic properties and enhanced potency.
In conclusion, the surface modified nanocrystals is
a potential novel drug delivery system with about
100% drug load and minimum carrier responsible
for specific, selective and targeted drug delivery.
ALNCD, dolutegravir nanocrystals, SILCRYST™
nanocrystals, Ryanodex and EquivaBone are few
18 Current Nanomedicine, 2020, Vol. 10, No. 1
Fig. (3). Surface modifiers to nanocrystals.
of the various nanocrystalline systems recently re-
ceived approval by the USFDA. Thus, the surface
modified nanocrystal can be a solution to the
pharmaceutical industry for drugs belonging to
BCS class II and IV by increasing its solubility,
stability, potency, permeation, and thus bioavaila-
bility.
9. PROSPECTS FOR FUTURE GENERA-
TION
Surface modified nanocrystals have gained tre-
mendous popularity attracting researchers towards
its associated advantages, ease of manufacturing
and positive results. Till now, targeted nanocrys-
tals have been reported by many researchers for
having an advantage over non-surface-modified
nanocrystals, reflecting its importance in drug de-
livery such as delivery to cancerous cells, brain
targeted drug delivery, ocular delivery, pulmonary
delivery, etc. In the future, multi-targeted or multi-
modal nanocrystals can provide the unmet de-
mands of current therapies of life threatening dis-
eases like cancer, neurodegeneration and com-
municable diseases. Also a comparative study of
targeted nanocrystals, multi-modal nanocrystals,
free drugs, and drugs in other nanocarriers, can be
performed to determine the actual extent of effec-
tiveness of nanoscale platforms.
CONCLUSION
Nanocrystals, currently are found to be the most
suitable and viable option for enhancing the aque-
Kumar et al.
ous solubility and thus bioavailability of the poorly
aqueous soluble drug. Nanocrystals can be dis-
cussed under three generations based on progress
and modification from its origin to the present.
While, the first generations simple nanocrystals
did not provide any targeted delivery and were
prone to elimination by the mononuclear phago-
cytic system (MPS), second generations nanocrys-
tals saw encapsulation or entrapment of nanocrys-
tals inside a delivery system to prevent its elimina-
tion by MPS and finally surface-modified third
generation nanocrystals, provided an option for
targeted delivery. It presented a carrier-free drug
delivery system with a maximum drug load and a
hydrophilic surface with a ligand attached to it,
solving the limitations of earlier generations of
nanocrystals as well as conventional nanocarriers.
CONSENT FOR PUBLICATION
Not applicable.
FUNDING
None.
CONFLICT OF INTEREST
The authors declare no conflict of interest, fi-
nancial or otherwise.
ACKNOWLEDGEMENTS
Declared none.
Authors Review on Drug Nanocrystals
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