Engineered Nanocrystal Technology: In-Vivo Fate, Targeting and Applications in Drug Delivery

Journal of Controlled Release.
2014, 183, 51-66
Engineered Nanocrystal Technology: in-vivo fate,

targeting and applications in drug delivery
Vivek K. Pawar, Yuvraj Singh, Jaya Gopal Meher, Siddharth Gupta, Manish K.
Chourasia*
Pharmaceutics Division, CSIR-Central Drug Research Institute, Lucknow, UP, India

*Corresponding author; Pharmaceutics Division, CSIR-Central Drug Research Institute, Lucknow, UP, India 226031. Fax:
+91 522 2623405; Tel: +91 522 2612411-18; E-mail: manish_chourasia@cdri.res.in
Abstract
Formulation of nanocrystals is a robust approach which can improve delivery of poorly water
soluble drugs, a challenge pharmaceutical industry has been facing since long. Large scale
production of nanocrystals is done by techniques like precipitation, media milling and, high
pressure homogenization. Application of stabilizers along with drying accords nanocrystals
long term stability and commercial viability. These can be administered through oral,
parenteral, pulmonary, dermal and ocular routes showing their high therapeutic applicability.
They serve to target drug molecules in specific regions through size manipulation and surface
modification. This review dwells upon the in-vivo fate and varying applications in addition to
the facets of drug nanocrystals stated above.
Keywords: Nanocrystals, Poorly Soluble Drugs, In-Vivo Fate, Bioavailability, Stability,

Targeted Delivery.
1. Introduction
With the advancement of drug delivering technology, use of engineered nanoparticles has
revolutionised the diagnosis and treatment of disease by targeting drugs to various cellular
sites. Previously, attaining such a selective delivery and reduced dose dependent toxicity
(especially in case of anticancer drugs) with conventional methods were dream merely [1].
Almost 70 % of the newly researched molecules are facing issues of poor aqueous solubility
which finally lead to poor bioavailability [2]. Many strategies have been adopted to overcome
this problem like surfactant utilization [3], inclusion complexation[4], co-solvency[5], solid
dispersion[6] etc., and emphasis is being laid on preparation of safe and effective dosage
forms with better bioavailability. Reduction of particle size to nano-metric region is also
being applied to achieve the same. Governed by Noyes Whitney theory, smaller particles
with grater surface to volume ratio (Fig.1) effectively increase the dissolution rate of active
ingredients by raising their saturation solubility[7, 8]. Nanocrystals can follow above
mentioned criteria because they are developed in a crystalline state which has particle size in
nano-scale[9-11]. Thus, nanocrystals enhance the bioavailability of drugs which undergo
erratic absorption[12-14]. They do not involve use of extreme pH ranges for solubilisation,
due to which it is possible to reduce the solvent related adverse effects. Another distinct
advantage of nanocrystals is their capability to offer almost 100% drug content which raises
the probability of obtaining higher therapeutic concentration producing desired
pharmacological action[15] and makes them superior to other colloidal drug delivery systems
(Fig. 2). They can be administered through various routes including parenteral[16],
ocular[17], oral [18] and pulmonary[19] which make them extremely versatile.
Thorough investigations on nanocrystals have been carried out in past 10-15 years which led
to development of many marketed products (Table 1). Various production technologies have
been brought in use for the development of these nanocrystals which can be summarised as:
bottom-up (precipitation), top-down (media milling and high pressure homogenization),
Journal of Controlled Release. 2014, 183, 51-66
combination approaches and chemical synthesis. Additionally, during the developmental

journey of nanocrystals certain modifications are implemented for obtaining desired
properties. High industrial scalability of production methods suggests that long unfulfilled
potential held by therapeutic nanotechnology has been duly realized by nanocrystals[20].
Considering the prospects and industrial consequences of nanocrystals, an effort has been
made to summarize different strategies used for formulating nanocrystals and challenging
issues in their long term stability. Furthermore, hypothesis/postulate expounding “in-vivo fate
of nanocrystals” aided with corresponding experimental findings substantiating these claims
has also been brought forward. Current perspective ends with detailed introspection into
targeting potential of nanocrystals in biological systems along with their versatile
applications in drug delivery.
Fig.1: Artistic impression depicting higher dissolution rates linked with particle size reduction. Intuitively,
associating smaller sized particles to larger surface area is difficult to comprehend. But as is self explanatory
from the diagram which employs cube as a model drug crystal, conversion into nanocrystals raises the surface
area massively for the same volume of drug. Exposition of new surface area (pink coloured) as a result of
reduced particle size is available for drug dissolution. This mechanism plays dominant role in raising the
intrinsic solubility of many water incompliant drugs.
Fig. 2: Structural arrangement of (A) Nanoparticles (B) Nanoemulsions (C) Liposomes and (D) Nanocrystals
with emphasis on drug distribution. Nanocrystals are unique as drug itself makes the bulk of carrier unlike other
colloidal carriers which consist of drug dispersed or entrapped in release rate controlling layer. Excipients
employed in formulating nanocrystals provide them with additional stability and/or targeting properties with
limited role in drug release. Shape and size of nanocrystals has special relevance in predicting their functional
outcome.
Table 1.Marketed products formulated using drug nanocrystal technology in healthcare

Product/company Active Indication Method of Route
Ingredient preparation
b
Invega Sustenna/ Paliperidone Antidepressant Top-down, HPH Parenteral
J&Ja palmitate
b
Megace®ES / Par Megesterol Appetite stimulant Top-down, HPH Oral
Pharma acetate
Cesamet®/Lilly Nabilone Anti- emetic Bottom up, co- Oral
precipitation
Tricor®/ Abbott Fenofibrate Hypercholesterolemia Top-down, media Oral
milling
Emend®/ Merck Aprepitant Anti- emetic Top-down, media Oral
milling
Rapamune®/ Wyeth Sirolimus Immunosuppressant Top-down, media Oral
milling
a
J&J- Johnson&Johnson, bHPH- High Pressure Homogenization
2. Fabrication Technologies
Bulk of nanocrystals is made up of drug itself with nominal utilization of excipients like
surface stabilising agents. Simplistic manufacturing technology and low ingredient
requirement reduce the overall cost of production making the process easy for scale up. They
can also be sterilized easily with conventional methods including heat/steam/radiation
sterilization. Scientists have explored various consequences of sterilizing nanocrystals and
come up with a completely sterile and healthy facility for producing nanocrystals of cytotoxic
drugs[21, 22]. Bottom up and top down technologies are the two basic approaches for the
production of nanocrystals and a combination of these two technologies is often sufficient to
induce size reduction in most of the cases (Fig. 3).
2.1. Bottom up technologies
Also termed as nanoprecipitaiton, Bottom up technique was first introduced by List et al [23].
This technique involves solubilisation of drug in a suitable solvent followed by precipitation
of dissolved drug via adding a non-solvent which results into production of nanocrystals [24].
However, most of the modern therapeutic entities are not easily soluble in common organic
solvents which themselves are toxic and difficult to completely remove thus limiting the
applicability of the method[25]. With expense of time, advancement in technologies has led
to production of nanocrystals using various supercritical fluid technologies. These
technologies involve gas anti-solvent recrystallization (GAS) , aerosol solvent ex-traction
system (ASES)[26], atomised rapid injection for solvent extraction (ARISE), rapid expansion
of supercritical solution (RESS)[27] and depressurization of an expanded liquid organic
solution (DELOS)[28].
2.2. Top down technologies
These technologies employ a high force for diminution of particle size and can be applied to a
wide range of insoluble ‘brick dust drugs’ that generally face solubility issues. A traditional
top down technology usually produces nano-size crystals by milling a drug. Initially, jet
milling was used for particle size reduction but was overtaken by wet milling because it
offers better possibilities for attaining nanonization. Wet milling is a classical technology that
involves a media milling chamber, a dispersion medium usually water and a suitable
stabilizer to achieve reduction of particle size. Wet milling utilizes both high and low energy
process depending upon the nature of particles. Pearl mill also called NanocrystalTM
technology is a low energy milling process and has produced many nano-sized commercial
products (Rapamune, Emend®, Tricor® and Megase ES®)[24]. Simultaneously, a Canadian
company RTP developed a high energy process, called as high pressure homogenization, with
the use of a microfluidizer which works on the principle of collision and cavitation. Later,
this technology was taken up by SKyePharma PLC, London, UK. Based on the same
principal, a technology, named Cubes®, was developed by Muller and co-workers. They
utilized piston gap homogenizer for size reduction. With progression in the field of
formulation technology, changes were brought to the classical approaches by partial or full
replacement of water as suspending media with oil, polyethylene glycol (PEG) or glycerol.
This change was made by Pharmasol (Berlin, Germany) during the development phase of
Nanopure® technology which allowed direct filling of nano-formulations into capsules or
injectables[29].
2.3. Combination technologies
The specific advantages of above mentioned technologies were amalgamated by Baxter
(Baxter Pharmaceutical Solutions, LLC, US) in form of NANOEDGETM which introduced a
pre-treatment step where the crystals are precipitated to produce a suspension followed by
high pressure homogenization. Baxter has modified these technologies to formulate an
injectable drug delivery system for poorly soluble drugs which remain stable but product
launching in the market still remains farfetched[7]. However, literature is loaded with
examples where combinational technologies have produced stabilized nanocrystals[30-33].
Fig. 3: Manufacturing techniques employed towards fabricating nanocrystals. Top down methods process
primary drug dispersions and scale down the size of drug particles by milling, cavitation, grinding, impaction,
shearing, attrition etc. Bottom up techniques work on the principle of nanoprecipitation in which drug solution is
introduced in a non-solvent system to initiate nucleation followed by size growth towards adequate size.
Stabilizers are often incorporated in formulation mixture to prevent size fluctuation. A combination of these two
traditional methods can also be applied to manufacture nanocrystals.
3. Nanocrystals Stabilization
Despite, offering an impressive array of advantages, the small size of nanocrystals can often
lead to stability concerns. The massive surface area of nanocrystals results in sufficiently high
free energy or surface charge that might cause attraction or agglomeration[34]. Small sized
nanocrystals sometimes raise the solubility of drug beyond the saturation point which
promotes recrystallization into larger particles; also known as Ostwald ripening. These
processes ultimately lead to irreversible loss of formulation integrity.
The simplest indicator of instability associated with nanocrystals is particle size growth.
Increment can be monitored by differential light scattering method under different sets of
conditions. Various advanced analytical techniques such as X-RD, DSC, NMR and FT-IR are
also employed to judge the stability of nanocrystals.[18, 35] In addition to formulation
behaviour during shelf life, detailed investigations should be carried out to validate the in-
vivo stability profile of nanocrystals. For instance nanocrystals intended for oral delivery
must be capable of withstanding the harsh gastrointestinal environment. Likewise, parenterals
are subjected to testing in physiological fluids. Furthermore, nanocrystals of water insoluble
drugs are always susceptible to precipitation upon dilution by gastric and other body fluids
after administration into the body[36].
Formulation based approaches have been adopted to check such instabilities. Ostwald
ripening can be negated by employing a suspending media in which the bioactive is
extremely insoluble. Agglomeration of nanocrystals can be curtailed by surface stabilisation
using a suitable amphiphilic stabiliser having both hydrophilic and hydrophobic domains in a
single functional molecule. The hydrophilic group is suitable for increasing the solubility of
poorly soluble drug whereas hydrophobic group accords improved stability to the suspended
particles in the dispersion medium[37]. A study demonstrated that hydrophilic compounds
having very low enthalpy value are less suitable stabilizer for development of nanocrystals
and their choice is dependent upon the hydrophobicity of the subjected drug molecule[38].
Some of the excipients used for stabilization of nanocrystals are summarised below:
3.1. Poloxamers
Poloxamers are amphiphilic block copolymers formed with a combination of ethylene oxide
(E; hydrophilic) and propylene oxide (P; hydrophobic) unit arranged in an E-P-E
arrangement. Poloxamers are available in various grades developed using different lengths of
polymer blocks. They exhibit versatile range of applications in drug delivery due their
multiple effects including solubility alteration, stability impartment, and reduction in protein
binding. They not only serve as ideal stabilizers but also presume the capacity to
chemosensitize the multiple drug resistance (MDR) cells [39], [40, 41]. Poloxamers are
certified as generally recognized as safe (GRAS) excipient and considerably cause negligible
haemolytic reaction hence they are popular for delivery of drugs through intravenous
route[42]. They have been widely used for the stabilization of nanocrystals[43-45].
Poloxamer F188 appended omeprazole nanocrystals showed enhanced stability due to
shielding of the compound and on comparison with omeprazole solution, they demonstrated
no discoloration in sodium bicarbonate solution[46]. A better stability approach has also been
made by Ganta et al during the formulation of ascularine nanocrystals where the hydrophobic
core of the polymer held the molecules stable by binding onto the particle surface[47].
Similar, tarazepide, a poorly soluble CCKa-antagonist, was nanomerized using combination
of poloxamer 188 with polysorbate 80 and glycerol. The developed formulations remained
unchanged over a period of three months however it was seen that glycerol only had a trivial
role in stabilizing tarazepide nanocrystals[48]. Owing to its ideal combination with
polysorbate 80, nanocrystal formulations bearing amphotericin B[49] and loviride[50] were
developed. Rabinow et al developed itraconazole nanocrystals using poloxamer 188 and
described their role in favourable manipulation of pharmacokinetic with decreased Cmax and
increased Tmax[51]. Mishra et al., fabricated hesperetin nanocrystals for dermal application by
employing four different stabilizers (poloxamer 188, polysorbate 80, Inutec SP1 and decyl
glucoside) and they observed that poloxamer 188 was more efficient in preserving the size of
developed nanocrystals[52].
Various researchers have also utilized poloxamer 407 for the development of several
nanocrystals formulations. Deng et al., attempted to improve the therapeutic profile of
paclitaxel by stabilizing its nanocrystals using poloxamer 407, but failed, and ended up with
thermo-sensitive miceller structure. However, renanonization with incubation sonication led
to formation of nanocrystals with prolonged stability[40].
3.2. Poly vinyl pyrrolidone (PVP)
PVP or povidone is prepared by reaction of acetylene and pyrrolidone to form vinyl
pyrrolidone followed by polymerization to convert into PVP. It is available in different
viscosity grades having a versatile range of application from being binder in tablets and
capsules, film formers in ophthalmic solution, taste masking agent, toxicity reducer and the
most important it works as stabilizer in suspensions.
PVP K30 has been applied as stabilizer for formation of celecoxib nanocrystals. Two nano-
formulations were made; one stabilized with a combination of PVP K30 with sodium dodecyl
sulfate (SDS) in a ratio of 1:1 and other with tween 80. Remarkably, it was seen that
combination of stabilizers did not affect the crystallinity of drug when characterized by DSC,
however a reduction of melting point was seen due to generation of new crystalline state[53].
PVP K17 and K12 demonstrated the versatile applications of PVP when they were tried for
the preparation of probucol nanocrystals. The study established the fact that PVP or SDS
alone was incapable to prevent agglomeration whereas combination of both resulted in a
stable formulation. In this work researchers used two different grades of PVP (K12 and K17)
in combination with SDS and found that a stable formulation was obtained with use of PVP
K17 and SDS in comparison to PVP K12 and SDS due to sufficient coverage of drug
surface[54]. Similarly, Douroumis et al., proved a blend of PVP K12 and PVP K17 with
hydroxypropylmethyl cellulose (HPMC) provided excellent stability to carbamazepine
nanocrystals. There was a remarkable improvement in the stability of the formulation which
contained PVP K17 in the stabilizer mixture[55].
3.3. Poly Vinyl Alcohol (PVA)
Properties of the water soluble PVA is dependent upon the degree of polymerization and
extent of hydrolysis. Partially hydrolysed PVA is generally used in pharmaceutical
industry[42]. It has been used in formulating stable nitrendipine (a class II calcium channel
blocker) nanocrystals through precipitation ultra-sonication method with resultant improved
dissolution characteristics which in turn increased its oral bioavailability. A total of six
combinations with varying concentration of PVA from 0.1 to 1.5% were formulated and
formulation containing 0.2% PVA was found to be sufficiently stable for over a period of six
months[56].
3.4. Amino acid derived co-polymers
Albumin, a single polypeptide chain of 585 amino acids, is generally used as stabilizing agent
for parenteral formulations containing protein and enzyme. Leucine (C6H13NO2) has gained
usage as lubricant and an anti-adherent in the development of aqueous nanocrystal
formulation. Lee et al., tried combination of various co-polymers derived from amino acids to
stabilize nanocrystals consisting of naproxem. Nano-formulations were developed using two
polymeric combinations made of lysine, leucine, and albumin. Out of these two
combinations, lysine (hydrophilic moiety) and leucine (hydrophobic moiety) was found to be
successful in achieving the required particle size and stability[42, 57]_ENREF_25.
3.5. Brij-78
Commonly, used as emulsifying, wetting and a permeation enhancing agent, Brij-78 is a non-
ionic surfactant containing polyoxyethylene alkyl ethers and also termed as Cremophor.
Nanocrystals obtained by processing oridonin were stabilized using Brij-78 by Gao et al.
However, its use is often associated with anaphylactic hypersensitivity reactions,
hyperlipidaemia, abnormal lipoprotein patterns, aggregation of erythrocytes and peripheral
neuropathy[58]
3.6. Lecithin
These are considered as a mixture of phosphatides with triglycerides, fatty acids and
carbohydrates. Due to their lipid content, they form an integral part of many nutritional
formulations. When used in pharmaceutical industry, they excel as stabilizer or emulsifiers.
Their physical forms may vary from being powders or semi liquids based on their free fatty
acid content and they also posses good absorption enhancing property[42]. Being derived
from natural sources (egg and soya), they find wide acceptance as stabilizer for variety of
drugs. Lecithin was used in combination with poloxomer 188 and HPMC to stabilize
amoitone B, an anticancer agent. Different batches were prepared with varying concentration
of stabilizers keeping zeta potential and size as optimization factors. Combination of
poloxomer 188 and lecithin (1:1) was found to produce a stable formulation. HPMC failed to
provide desired stability since it did not adopt dual stabilization mechanism of steric and
electrostatic repulsion as executed by lecithin[59]. In a study elsewhere, twenty different
stabilizers were screened preliminarily to formulate curcumin nanocrytstals, amongst which
lecithin displayed best suspending capability along with its combination with SDC.
Maximum steric and electric repulsion offered by the blend of stabilizers were responsible for
stabilization of nanocrystals[60]. Lecithin has also been applied for development of RMKP
23 (an antibacterial compound), prednisolone and carbamazepine bearing nanocrystals[61].
Yang et al., have employed dipalmitoyl phosphatidylcholine (a lecithin; endogenous
component of human lung surfactant) to formulate nebulized itraconazole nanocrystals with
improved bioavailability. The presence of dipalmitoyl phosphatidylcholine ultimately
improved the overall in-vivo presence of itraconazole due to its permeation enhancement
property[62].
3.7. D-a-Tocopheryl polyethylene glycol 1000 succinate (Vitamin E TPGS / TPGS 1000)
Vitamin E TPGS an FDA approved water soluble compound is a waxy liquid and produced
by esterification of vitamin E with PEG 1000. Apart from being a very good emulsifier, it
also has P-glycoprotein (P-gp) inhibiting property. It is being increasingly employed to
overcome bioavailability issues associated with poorly soluble compounds especially in case
of anticancer drugs[63] where it helps to overcome the MDR. The benefits of TPGS have
been used in the form of sole surfactant or stabilizer in formation of paclitaxel nanocrystals
against MDR cells[64].
It has served as an optimal stabiliser in many formulations like baicalin (flavone)
nanocrystals where it is used along with SDS, HPMC, poloxomer 188 and methyl cellulose.
Out of all baicalin nanocrystals, the batch manufactured using TPGS showed the minimum
mean diameter (0.513 µm) and maximum redespersibility following lyophilization. Here, the
fully extended polymer chain of TPGS efficiently counterbalanced the stresses induced
during freeze drying and ultimately prevented particle agglomeration[65].
NVS-102 (a compound synthesised by Novartis Pharma) was converted into its
nanocrystalline form using TPGS as a stabilizing agent. NVS-102 nanocrystals were
produced using wet media milling technique and the effect of vitamin E TPGS on size and its
oral absorption was investigated. Encouraging results depicted by narrow size distribution,
increased absorption rate, extended stability and controlled Ostwald ripening showcased the
feasibility of TPGS stabilized nanocrystals[66].
3.8. Polysorbate 80
A polyoxyethylene sorbitan fatty acid ester derivative has established itself as an important
pharmaceutical excipient. They are classified according to type of fatty acid moiety which
influences their functions. It has been utilized on the large scale as surface active agent
however its propensity towards causing hypersensitivity[67], birth weight reduction in
infants[68] and other side effects when used in high concentration can some time be a
deterrent. Therefore, researchers often tend to utilize polysorbate 80 at low levels as a
complimentary stabilizer. It has been tried in combination with poloxomer 188, PVA, PVP
and SDS for eveloping fenofibrate nanocrystals[69].
3.9. Sodium lauryl sulphate
A sulfuric acid monododecyl ester sodium salt, is an anionic surfactant used widely as
wetting agent however it exerts moderate toxic effects including irritation of eyes, skin and
stomach. It has been used to developed nanocrystals of herpetrione, an antiviral agent
extracted from herpetospermum caudigerum, via high pressure homogenization technique
followed by freeze drying. The powder X- ray diffraction studies revealed that there was no
interaction or alteration in the true nature of drug by the stabilizer and an overall increase in
the oral bioavailability was reported[70].
3.10. HPMC
It is a widely used GRAS listed pharmaceutical excipient that has found special use in
formulation of nanocrystal and is believed to cover the surface of crystal efficiently providing
sufficient stability. In comparison to other stabilizers, its relatively high melting point makes
it robust option for production methods which involve high processing temperature. Recently,
Ali et al., utilized HPMC as a stabilizing agent in media milling process of hydrocortisone
nanocrystals intended for ophthalmic delivery. HPMC played a key role in stabilizing the
nanocrystals by completely covering the surface of dried particle and gave it a low zeta
potential. When combined with polysorbate 80 and PVP it rendered stability to the
formulation for a period of more than 2 months[31]. In another study, Figueroa et al.,
prepared HPMC based fenofibrate, naproxem, griseofulvin (BCS class-II drug) nanocrystals.
Inferences showed that HPMC was able to maintain the crystallinity of the bioactives[71].
Hecq et al, used HPMC for producing nanocrystals of nimodipine using high pressure
homogenization. The technology was utilized with the aim of formulating nanocrystals to
improve the dissolution rate of nifedipine. The use of low viscosity grade HPMC provided
adequate stabilizing effect compared to other surface active agents like SDS, poloxomer and
polysorbates[72].
3.11. Sodium cholic acid
Bile acid derived white crystalline powder is employed to stabilize many nanocrystals based
formulations. In combination with poloxomer 188, it has been used to stabilize nimodipine
nanocrystals. Nimodipine is regarded as a drug of choice for reducing both morbidity and
mortality in subarachnoid haemorrhage related vasospasm. The clinically available injectable
form of drug is administered with alcohol and PEG 4000 which is associated with numerous
allergic reactions. The sodium cholic acid stabilized nanocrystals thus offered a novel
approach in which the excipients did not adversely affect the risk benefit ratio and open an
option for intravenous administration of nimodipine[73]. It has also been used for
stabilization of cyclosporine nanocrystals[74].
4. Ensuring long term stability: drying

Despite, employing myriad of stabilizers and their intricate combinations complete preclusion
of crystal growth is unavoidable. The interplay of thermodynamics and molecular kinetics is
accelerated in presence of a liquid dispersion medium. Thus, drying of the liquid nanocrystals
formulation is often required in conjunction with stabilizers to obtain long term stability.
Removal of the liquid dispersing the nanocrystals can be attained by two types of drying
process: freeze and spray[75, 76]. These dried crystals so obtained can be processed into
suitable solid dosage form like tablet or capsule[77, 78]_ENREF_72. Care must be taken to
ensure that the rapid dissolution characteristics of the nanocystals must not be affected during
above said processing. In no case, wetting and disintegrating properties of the developed
solid dosage should play rate limiting role in the sequence of events which dictate the
dissolution of nanocrystals[79]. Also, incorporation of nanocrystals in the tablet dosage form
is limited to low dose drugs since higher dose can cause aggregation of nanocrystals within
the tablet matrix. With reference to freeze drying, the type and amount of cryoprotactant
plays a crucial role in maintaining the highly sensitive structural features of nanocrystals.
Absence of cryoprotactant leads to formation of non-frozen liquid micro phase resulting in
phase separation into ice and cryo-concentrated solution. This causes selective segregation of
nanocrystals in the small non-frozen liquid pockets increasing the probability of particulate
aggregation. Considerable amount of stress observed during freezing and dehydration further
destabilize the colloidal system. Therefore matrix formers like soluble sugars (sucrose,
glucose, mannitol, trehalose etc.) are added to the formulation before carrying out freeze
drying which cause immobilization of the nanocrystals thereby forming a protective shield
capable of withstanding mechanical stress and aggregation[80].
5. Fate of nanocrystals in biological environment

From many years, emphasis has been laid onto the benefits garnered from nanotechnology
however nano-toxicity related issues have also simultaneously cropped up[81]. Several
studies reported possible entry of nano-particulate formulations into individual cells
hindering the immune system[82-84]. Therefore, tracing the in-vivo movement of
nanocrystals becomes exceedingly important. Fate of nanocrystals in biological environment
is a controversial topic, as a wide spectrum of reports is available in this regard. Some groups
of investigators have claimed involvement of specialized transports for nanocrystals whereas,
others prioritize solubilisation followed by absorption of hydrophobic drugs fabricated as
nanocrystals[85, 86]. Thus, providing a concrete answer to the query “what exactly happens
to nanocrystals in biological environment?” is a tricky debate.
The primary concern about the fate of nanocrystals is route of administration as it ultimately
governs the sequence of events through which nanocrystals transpire to reach their active site.
Nanocrystals are expected to behave differently when introduced directly into five litres of
turbulence free blood circulation and bypassing absorption barriers, varying pH, peristaltic
movements, and other modalities associable with oral delivery.
Anecdotally nano-crystallization technology has been employed to improve intrinsic
solubility of bioactives, the level of which (solubility increment) often remains undetermined.
As, Fu et al., found out that nimodipine (poorly water soluble drug) nanocrystals were
completely soluble at 2 µg/mL whereas only partial solubility was attained at 200 µg/mL in
the same form [87]. Expectantly they concluded that molecular dispersion of nemodipine
obtained from solublized nanocrystals underwent classical passive diffusion leaving the
scope of specialized absorptive pathways for absorption of the remaining intact nanocrystals
which did not undergo dissolution.
Despite sharing several traits with other colloidal carriers, nanocrystals do not
necessarily adopt similar transport systems [88]. Researchers have reported that nanocrystals
are preferably absorbed from jejunum instead of ileum which signifies that a distinct
absorption mechanism is probably involved in their translocation [30].
Fig. 4(A) Graphical representation of intracellular tight junction and failure of nanocrystals to pass through
paracellular pathway, (1) enterocytes, (2) intracellular tight junction (< 10 Å), (3) failure of nanocrystals to pass
through the paracellular pathway
Paracellular pathway might be one such route for potential uptake of nanocrystals. However
owing to the strict diametric constriction (< 10 Å) of the pores present in between juxtaposed
cells, exponentially larger nanocrystals (~ 100 nm onwards) find it difficult to pass through.
Fig 4A illustrates paracellular pathway of enterocytes which hinders transport of
nanocrystals. However, some researchers claim the opening of these junctions by the
application of some specific chemicals (surfactanants) such as sodium deoxycholate which
might facilitate the entry of nanocrystals, but there is no adequate experimental evidence on
this[89]. In case of cancer the paracellular uptake at site of action may take place owing to the
well known enhanced permeability and retention (EPR) effect (high fenestra frequency) [90].
Fig. 4 (B) Diagrammatic representation of clathrin-mediated, non-clathrin (caveolae) mediated endocytosis of

nanocrystals after oral administration, (1) nanocrystals on the enterocyte surface, (2) magnified view of
nanocrystals over the cell membrane, (3) clathrin-mediated phagosome and endocytosis of nanocrystals, (4)
release of nanocrystals to the other side of cell via clathrin-mediated transport, (5) non-clathrin (caveolae)
mediated endocytosis of nanocrystals, (6) release of nanocrystals to the other side of cell via non-clathrin
(caveolae) mediated transport
Some nanocrystals are taken up directly by cells via multiple endocytotic pathways including
clathrin-mediated, non-clathrin mediated (caveolae), macro-pinocytosis, as well as
phagocytosis. Fig 4B demonstrates the above specialized mechanisms of nanocrystals
transport via enterocytes. In order to investigate the clathrin-mediated endocytosis of
nanocrystals Zhang et al., employed chlorpromazine (known to disrupt clathrin and thereby
clathrin-mediated endocytosis) in cellular uptake studies [30]. They found a significant
decrese in the uptake of nanocrystals which supports their hypothesis of clathrin-assisted
endocytosis. In continuation of their investigation Zhang and his fellow researchers explored
other possible pathways and found that cellular uptake of nanocrystals was lesser at 4°C in
comparison to 37°C, which might be attributed to the fact that endocytosis of nanocrystals
(which requires energy sources; ATP) might have been diminished at lower temperature.
These experimental findings support the uptake of nanocrystals via active membrane
transport process, most likely clathrin- and caveolae-mediated endocytosis. After endocytosis
nanocrystals possibly become available for lymphatic transport instead of venous transport.
The reasons might be the low pore size (roughly 3 nm) of the vascular epithelium. Reports on
the uptake of nanocrystals via M-cells are also found in literature. These specialized cells are
believed to transport nanocrystals through an active transepithelial vesicular transport system
from the lumen directly to lymphoid cells and tissues; though the overall efficiency of this
process remains questionable as M cell populate less than 1 % of the intestinal epithelia[91].
Following intravenous administration, naocrystals may remain intact or enter into a
molecular dispersion due to high instrinsic solubility, dissolution rate, rapid dilution
accessing the site of action for therapeutic effect [92]. Alternatively intact nanocrystals keep
moving in the systemic circulation as colloidal particles. During their flight, nanocrystals are
recognized as foreign bodies and engulfed by the defence (phagoctic) cells such as
macrophages. In phagocytic cells nanocrystals dissolve slowly in phagolysosomes.
Consequently, the hydrophobic drug might pass through the phagolysosomal membrane and
enter into the cytoplasm, and then diffuse out of the cell down its concentration gradient[93].
Fig 5 depicts the fate of nanocrystal after intravenous administration. Nanocrystals of size
less than 100 nm behave like molecular solution whereas those greater than 500 nm are
accumulated in liver. This characteristic of nanocrystals can be successfully used in targeting
different diseases/disorders [94].
To summarize nanocrystals and their uptake is not fully understood although several studies
have reported various mechanisms as well as pathways. More detailed in vivo experiments at
pre-clinical measure are required for the complete elucidation of mechanisms involved in the
in vivo fate of nanocrystals.
Fig 5 Proposed in vivo fate of nanocrystals after intravenous administration (1) injection of nanocrystals to
blood vessels, (2) nanocrystals in the blood stream in molecular solution and intact nanocrystals form, (3)
phagocytic cells, (4) formation of molecular solutions of nanocrystals in blood, (5) phagocytosis of intact
nanocrystals and further solubilization in cytosol of phagocytes, (6) drug in molecular solution form comes out
due to concentration gradients of phagocytes and outer environment
6. Applications
6.1. Oral drug delivery
Oral route has been the most preferred route and is considered as the safest and suitable route
for drug delivery[95]. For orally administered drugs, dissolution is considered as rate
determining step for absorption[96]. Nanocrystals provide a greater surface area for
dissolution and thus raising the saturation solubility which ultimately increases the
dissolution rate thereby enhance drug absorption. Rapamune® was the first USFDA approved
oral nanocrystals launched in the year 2000 by Wyeth Pharmaceuticals (Madison, NJ). It
consisted of sirolimus nanocrystals incorporated in an excipient mixture suited for direct
compression into palatable tablets. The oral bioavailability of the nanocrystals tablets was
21% higher compared to sirolimus solution[97].
The various advantages of nano size have been used to favourably modulate pharmacokinetic
profile of aprepitant (EMEND®) which follows absorption window phenomenon in the
gastrointestinal tract[98].
Nanocrystal technology has been known to even-out discrepancies between fasted and non-
fasted bioavailability of fenofibrate which is practically insoluble in water. Normally it
requires bile and related surfactants secreted postprandially for absorption. Nanomerisation of
fenofibrate enhances its solubility making it bioequivalent in fed and fasted condition[99].
Muller et al., have refined oral delivery of thermo stable drugs utilizing melted PEG (melting
point 60 ○C) which allows fixing of nanocrystals in a solid PEG matrix. Nanocrystals
dispersed in melted PEG were milled to powder and directly compressed into the tablet or
filled in capsules shell[29]. Thus, this novel drug delivery system offers a way to incorporate
poorly soluble drugs directly into tablet, capsule or hot melts solid matrix to improve oral
bioavailability.
6.2. Intravenous Drug Delivery
Administering a drug via intravenous route provides numerous benefits such as immediate
action, reduced dosing and 100% bioavailability. These benefits can be considered as ideal
parameters for every drug but the use of intravenous route is limited because harmful solvent
and excipients, which are used during formulation development, are also co-administered
with the drug and they can cause serious side effects other than the drug itself[51, 100].
Under such circumstances, nanocrystals could be considered as the ideal candidates for
intravenous delivery because their developmental processes do not employ excess use of such
harmful excipients. During development of a novel drug delivery system to be delivered
through parenteral route, it must be kept in mind that the carrier system should not be
phagocytosed by reticuloendothelial system as well as by Kuppfer cell present in the liver.
Thus, size range of ≤100nm is preferred for parentral nanocrystals[7]. Nanocrystals of drugs
such as ascularine[47], melarsoprol[16], oridonin[58], itraconazole[51], and curcumin[60]
have been successfully developed thereby providing benefits like increase in Cmax, and AUC.
These benefits are an open invitation to pharmaceutical scientists for exploiting nanocrystals
comprising water insoluble notorious drugs for intravenous delivery.
6.3. Pulmonary Drug Delivery
Lungs are highly perfused organs with a fully expanded surface area roughly equivalent to
three football fields. With no hepatic portal drainage, molecular dispersion of drug transport
rapidly with high efficiency into the systemic circulation. Recently, it has been demonstrated
that pulmonary nanocrystals have the ability to rival pharmacokinetics offered by intravenous
administration of baicalein[101]. Pulmonary route thus comes across as a viable option for
delivery of therapeutics. Due to constant exposure to external environment, it is highly
susceptible to disease causing agents; allergens and pathogens easily invade through
respiratory tract[102]. Conventional modalities of deep lung drug deposition have been
modified by tailoring size of nanocrystals. A nebulizer is generally required to administer
powdered nanocrystals. Nebulizer can incorporate nanocrystals into small inhalable droplets
(1-5 µm)[103]. A study showed that aerosolised nanocrystals enhanced drug bioavailability
when compare to inhalable dispersions containing microparticles[104]. However,
nebulisation is a complex process which depends upon variety of factors like droplet size and
breathing pattern. The above technology has been used to deliver budesonide through
pulmonary route.[19]
6.4. Ocular Drug Delivery
Ophthalmic drug delivery is a challenging task owing to critical pharmacokinetic
environment and physiological barriers of the eye that hinder the delivery of drugs[105].
Most of the drugs for ocular therapy are delivered through a topical formulation in form of
solution or suspension[106]. Conventional formulations are subjected to rapid clearance from
application site due to rapid eye movements (blinking) and lacrymation, which results in low
ocular availability. Short retention time of medication induces a need of repeated dosing
relatable with loss in patient compliance and dose dependent side effects. To alleviate these
rapid filings, several approaches like ocular inserts and opthalmic gels have been tried, which
themselves are associated with fair share of inadequacies, viz. poor therepeutic outcome,
blurred vision and local irritation. Ophthalmic drug delivery was believed to be benefited
largely by a colloidal drug delivery system. Piloplex, the first novel colloidal drug delivery
system developed, contains pilocarpine which is ionically bounded to poly (methyl)
methacrylate-co acrylic acid nanoparticles[107]. Subsequently, nanocrystal technology played
an advanced role in ophthalmic drug delivery tackling dispersibility issues of poorly soluble
drugs such as budesonide, dexamethasone, hydrocortisone prednisolone[17] and
fluorometholone[108]. Ali et al., used combination technology based upon microfluidic
nanoprecipitation and wet milling to create nanocrystals of hydrocortisone and ocular
bioavailability was evaluated in albino rabbits. Results demonstrated an extended duration of
action and significantly improved AUC of developed nanocrystals in comparison to free
drug[31]. A markedly advanced ophthalmic delivery system for forskolin (intra ocular
pressure lowering agent) was developed by incorporating its nanocrystals into an in-situ
gelling system comprised of poloxamer and polycarbophil. Pharmacodynamic studies
revealed that nanocrystals/hydrogel system efficiently lowered the intraocular pressure upto
12 h in comparison to conventional suspension[109].
6.5. Dermal Drug Delivery
Skin is a therapeutic barrier, limiting delivery of many drugs[110]. Success in dermal
delivery depends upon the permeation of drugs across stratum corneum[111, 112]. Due to
their small size, nanocrystals are expected to pack closely to form an occlusive layer which
hydrates the skin increasing penetration and permeation of drugs. Dispersed nanocrystals are
retained topically for sufficient time period, slowly releasing the active constituent[113].
They have already been tried with L- ascorbic acid to form a long term stable topical
formulation and containing nanocrystals in oil base[114]. Suspended nanocrystals of
lipophilic compounds like lutein, an antioxidant, showed an increased permeation through a
synthetic membrane[115]. Table 2 enlists some of the drugs which have been successfully
administered through various routes as nanocrystals.
Table 2 Drug delivery routes explored by nanocrystals

Drug Manufacturing Mean Use/ benefits Reference
technique particle size
Oral delivery
Danazol Media milling 169 nm Hypoestrogenic and hyperandrogenic activity/ a [18]
16 fold increase in bioavailability in comparison
to danazol suspension
Ketoprofen Media milling 265 nm Rheumatoid arthritis/ 1.2 fold increase in Cmax and [116]
2 fold reduction in Tmax in comparison with
microcrystalline ketoprofen
Fenofibrate HPH 356 nm Lipid lowering agent/ 12.5 fold increase in Cmax [117]
and 17 fold increase in bioavailability
Cyclosporine HPH 962 nm Autoimmune disease [74]

Itraconazole Precipitation 267 nm Antifungal/ 1.5 and 1.8 fold higher bioavailability [118]
from commercial product in the fed and fasted
states
Icaritin Antisolvent- 220 nm Prevent osteoporosis/faster dissolution, improved [119]
crystallization absorption and grater in-vivo bioactivity than raw
method suspension
under ultrasonication
Paraterphenyl Precipitation 200 nm Promising anticancer agent/ increased saturation [120]
derivative followed by HPH solubility, accelerated dissolution and 5 fold
higher AUC with significantly longer MRT in
comparison to its solution
Cilostazol Anti-solvent and 326 nm Vasodilator/enhanced AUC and Cmax was [121]
high-pressure observed in comparison to marketed formulation
homogenization
method
Intravenous delivery
Oridonin HPH 103.3 nm Anticancer/ improved bioavailability in [122]
Ascularine HPH 133 nm Anticancer / 2.3, 6.2, 2.7 and 2.7 fold increase in t [47]
1/2, Vd, CL, and MRT in comparison to ascularine
solution
Curcumin HPH 210.2 nm Anticancer / 3.1 fold increase in Cmax, 11.2 fold [60]
increase in MRT and 4.8 fold increase in AUC
Flurbiprofen HPH -- Rheumatoid arthritis/improved bioavailability [123]
Atovaquone HPH 279 nm Improved activity against toxoplasma encephalitis [124]
due to enhanced bioavailability
Pulmonary delivery
Itraconazole Precipitation Less than 1 Antifungal [125]
µm
Budesonide --- Less than 1 Anti-asthmatic [126]
µm
Buparvaquone --- --- Antiprotozoal [127]
Sildenafil Precipitation --- Erectile dysfunction and pulmonary hypertension [128]
Carvedilol Solvent 190 nm Antihypertensive [45]
precipitation-
ultrasonication
method
Ocular delivery
Hydrocortisone Precipitation 300 nm Steroid / 1.8 fold increase in AUC _ENREF_65
[31]
Forskolin Wet milling 164 nm Antiglaucoma agent/ improved intraocular [109]
pressure lowering efficacy than its solution form
Cyclosporin A In-situ precipitation 505 nm Immunosuppressant/improved bioavailability [129]
Mycophenolate HPH 440 nm Immunosuppressant/modified corneal drug [130]
mofetil disposition
Dermal delivery
L-Ascorbic acid Emulsification + 148 nm Antioxidant / long-term stable topical [114]
homogenization formulation without decomposition
Lutein HPH 429 nm Antioxidant [115]
Hesperetin HPH 300 nm Antioxidant and antiallergic [52]
Tretinoin Precipitation 324 nm Anti-acne agent [131]
Ibuprofen Wet milling 284 nm Analgesic [132]
7. Targeted nanocrystals
Nanotechnology has dramatically re-shaped drug delivery in recent years. It has motivated
researchers and industrialists to alter methods of formulating new as well as existing drugs
extending their lifespan in the process. Unscrupulous drug administration for treating a
localized disease is no longer acceptable. The extensive adverse effects caused by
chemotherapeutics intended for benign tumours are one such example where the entire body
bears the brunt of non-selective drug distribution. It therefore becomes exceedingly important
to design mechanisms which ensure targeted delivery. Targeting of drug molecules against
diseases requires careful consideration of their physicochemical and biopharmaceutical
properties and it can improve the current therapeutic profile of various active pharmaceutical
ingredients.
Targeting approach has also been implemented with nanocrystal technology for drugs which
frequently face challenges like bioavailability, solubility, stability and poor IVIV correlation
[133] [134]. Bupravaquone nanocrystals suspended in a mucoadhesive system were used for
targeting Cryptosporidium parvum, a gastrointestinal parasite. The nanocrystals showed a
better targeting and were considerably more stable than pure bupravaquone[135]. Surface
modification of atovaquone nanocrystals with surfactants like SDS was reported to enhace its
brain uptake and efficacy to derease parasite load (Toxoplasma gondii) in comparison to
commercial micronized atovaquone suspension (Wellvone®) oral administration of 100 mg of
drug/kg[136]. In a recent study, poorly soluble anti-retroviral nevirapine was converted into
nonocrystals using cold HPH followed by surface modificaiton with serum albumin, dextran
and PEG. The rank order found for macrophage (residance site of HIV) upkake was as
follows: serum albumin coated > dextran coated > PEG coated > uncoated nanocrystals >
free drug. Formulation coated with serum albumin showed atleast four fold higher
macrophage uptake then free drug. Biodistribution studies in rats for nanocrystals revealed
increased drug accumulation in brain, liver and spleen in comparison free drug when
administered intravenously[137]. Surface modification of fabricated nanocrystals can bring
the site specific targeting. Surface labelling of nanocrystls with fluorescent markers has been
widely utilized for diagnostic consequnces for example Williams et al., modified the surface
of silicon-substituted hydroxyapatite nanocrystals through a silane followed by grafting
fluorescein-5-maleimide to accommodate real-time biological cell trafficking.
Hydroxyapatite nanocrystals residence in pre-osteoblast cells was confirmed by observing
fluorescence upto 24 h without quenching. It was also forcasted that developed nanocrystals
would open the door for successful intracellular delivery of growth factors[138].
Cancer presents yet another opportunity for exploiting targeted drug delivery by nanocrystals
to tilt the risk benefit ratio towards improved therapeutic outcome [139]. The micro
environment surrounding a tumor is unique and not evidenced in normal vasculature which
can be characterized by narrow junctions between endothelial cells, ordered cellular
arrangement and adequate lymphatic drainage. However, vessels in and around tumor are
characterized by irregular endothelial cell lining, lack of pericytes, an indecorously developed
smooth musculature with randomly dispersed collagen. This improper arrangement of
endothelial cells leaves gaps of micron range in the tumor vasculature which can be easily
permeated by nanocrystals. Contribution of hypoxia and acidosis was also seen due to
disturbed microvasculature[140]. Additionally, lymphatic vessels are either absent or
ineffective causing inefficient drainage from the tumor tissue leading to effective retention or
accumulation of the drug carriers (EPR effect). The EPR effect/passive targeting also
prevents undue toxicity by providing selective localization of drugs[141] (Fig. 6).
Nanocrystals with their unique size achieve a new biodistribution profile which is not
possible with simple micronization of drug molecules. Zhang et al., whilst investigating the
in-vivo anti-tumor effect of camptothecin nanocrystals in MCF-7 xenografted Balb/C mice
observed significant tumor suppression due to their fivefold higher tumoral accumulation in
comparison to camptothecin solution. They attributed enhanced efficiency of nanocrystals to
EPR effect[30]. Different studies demonstrated that oridonin nanocrystals were found to
show enhanced apoptotic activity in prostatic PC-3 cell line and SMMC-7721 cells along
with better in-vivo tumor inhibition profile in comparison to oridonin solution[142-
144]_ENREF_112_ENREF_110. In another study, soya lecithin stabilized docetaxel nanocrystals
were prepared using HPH. The in-vivo pharmacokinetics, tissue distribution as well as anti-
tumor studies in B16 melanoma bearing Kunming mice suggested that intravenous
administration of docetaxel nanocrystals could effectively ameliorate tumor growth and
reduced toxicity by increasing accumulation of docetaxel within tumor sites in comparison to
marketed product[145].
Fig 6. EPR effect; (A) Normal vessel: the narrow gap junctions present in between
endothelial cells allow only small molecules to penetrate, screening out colloidal sized
particles. Notice the ordered structure of cells in presence of functional lymphatic drainage.
Lymph flow regularly filters out accumulated material. (B) Tumor microenvironment: The
vascular endothelium in and around tumor is disjointed, irregular and leaky allowing
effective penetration of nanocrystals. Absent or dysfunctional lymphatic vessels further
delays clearance of these particles leading to their enhanced accumulation at tumor site
Resistance to therapy is an adaptive strategy undertaken by the cancer cells to nullify the
advantages acquired through passive targeting. Structural incongruity is never consistent
leading to altered permeability and blood supply. To overcome such hurdles and, sometimes
to augment passive targeting, ligands selective for receptors over expressed on cancer cells
are appended to nanocrystals. This opens up the avenue for active receptor based targeting by
tailoring nanocrystals[146]_ENREF_112_ENREF_110. Liu et al., described the concept of proof
by activating paclitaxel nanocrystals through application of folic acid conjugated stabilizer. In
comparison to non-ligated paclitaxel nanocrystals, ligated nanocrystals showed enhanced in-

vitro cytotoxic activity due to preferential uptake via overexpressed folic acid receptor on KB
cells[147]. In another study, folate receptors were targeted using PTK75 nanocrystals
stabilized with poloxmer conjugated to folic acid and in-vivo studies indicated 10 fold
increased accumulation of drug in tumor[148]. An attempt was made to shed further light on
the detailed mechanism elucidating active cancer targeting by Huang and associates. They
carried out quantitative tumor uptake studies on PEG stabilized columnar gold nanocrystals
covalently attached to targeting peptides responsive to receptors selectively populating the
cancer cells. Gold nanocrystals were conjugated to three different ligands, namely: a single-
chain variable fragment which attaches to epidermal growth factor receptor; amino terminal
fragment peptide which acts on the urokinase plasminogen activator receptor; and cyclic
RGD peptide capable of binding on integrin receptor. Although the results of study revealed a
marginal increment in drug accumulation in comparison to non-targeted nanocrystals, but
they could greatly influence the cellular uptake of gold; illustrating how active homing device
can synergise with passive size based targeting in cancer therapy[149]. The space of actively
targeted nanocrystals is still void and needs to be carefully followed to produce commercially
successful products. Various diseases approached by nanocrystals are summarised in Table 3.
Table 3 Various disease states targeted by nanocrystals

Disease Drug Mfg. technique Route Benefits Referenc
e
Melarsoprol HPH i.v. Improved in-vitro antileukemic activity [16]
in comparison to its hydroxypropyl-β-
cyclodextrin inclusion complex
Hydroxy precipitation- i.v. Nanocrystals showed higher in-vitro [150]
camptothecin combined HPH cytotoxicity against cancer cells than
its injection form
Paclitaxel Precipitation i.v. Significant anti-tumor efficacy of [64]
followed by nanocrystals over taxol.
sonication
Oridonin HPH i.v. Improved in-vitro cytotoxicity and in- [142]
vivo antitumor activity were obtained
in comparison to oridonin solution.
Paclitaxel & 3-phase Oral & i.v. Enhanced antitumor activity due to [147]
camptothecin nanoparticle targeting folate receptors
engineering
technology
Oridonin HPH - Improved cytotoxicity and apoptotic [143]
Cancer activity against PC-3 cell line in
Docetaxel HPH i.v. Docetaxel nanocrystals proved a better [145]
opportunity to inhibit tumor growth
and reduce toxicity in comparison to
marketed formulation.
Camptothecin Precipitation i.v. Prepared nanocrystals had 5 times [30]
followed by more concentration in tumor compared
sonication to drug solution with a proven stability
for six months.
Oridonin HPH i.v. Effective inhibition of SMMC- 7721 [144]
cells was found using oridonin
nanocrystals and further confirmed by
higher in-vivo anticancer activity in
comparison free oridonin
Silybin HPH --- Low bioavailability and poor aqueous [151]
solubility limited the use of silybin as
an anticancer agent. Use of nanocrystal
technology lead to development of a

formulation which showed higher
anticancer activity against prostatic
cancer than the silybin solution.
Bicalutamide Anti-solvent Oral Nano sizing showed a significant [152]
Precipitation improvement in pharmacokinetic
profile of the drug after oral
administration indicated by 3.5 times
raised Cmax and AUC in comparison to
free drug.
2- Nano i.v. Nanocrystals showed significantly [153]
methoxyestradiol precipitation better tumor inhibition activity than its
(2- ME) high frequency solution form.
ultra-sonication
Amoitone B HPH Oral The formulated nanocrystals led to an [59]
increased dissolution velocity and
magnified AUC compared to amoitone
b solution.
Puerarin HPH i.v. Puerarin nanocrystals showed a better [154]
in-vivo tolerability and higher
anticancer efficacy than its free
solution.
Riccardin D Combination - Nanocrystals exhibited improved [155,
technologies solubility and dissolution profile. 156]_EN
Particle size affects the REF_154
pharmacokinetics and biodistribution
of nanocrystals. Big particles were
uptaken by reticuloendothelial system
at high level than smaller particle.
PIK75 HPH - 11 fold improvement in saturation [148]
solubility
SKLB610 Wet media oral 2.6 fold higher bioavailability was [157]
milling observed in comparison to simple
suspension of drug
HIV Nevirapine HPH i.v. The nano sizing resulted in better [137]
/AIDS/ accumulation of drug in different
Anti- organs of rat compared to plain drug
viral evidenced by higher MRT values in
brain, liver and spleen.
Rilpivirine wet milling i.m./ s.c. Results demonstrated that size of [158]
nanocrystals significantly influence the
pharmacokinetic profile of drug.
Nanocrtstals having size around 200
nm showed higher plasma drug
concentration in comparison to
nanocrystals having size 400 and 800
nm.
Efavirenz Media melling Oral Nanocrystals provide improved [159]
bioavailability due to enhanced
solubility, dissolution velocity,
permeability and absorption in
comparison to marketed formulation.
Herpetrione HPH Oral Significant higher AUC and Cmax with [70]
improved efficacy in comparison to
coarse drug suspension
Hemor Diosmin Precipitation --- 89% drug permeated from intestine in [160]
rhoids ex-vivo study
and
venous
leg
ulcers
Malari Atovaquone Microprecipitati Oral 4.6 and 3.2 fold higher AUC in [161]
a on with HPH comparison to drug suspension and
marketed formulation
Fungal Nystatin Wet media Intra oral Lowered oral burden of Candida [162]
infectio milling albicans with no systemic absorption
ns
Organ [163]
Targeti Nanocrystals coated with polysorbate
ng Amphotericin-B HPH 80 and sodium cholate showed
(Brain) enhanced brain uptake than
amphotericin b solution.
Atovaquone HPH Oral An approach was made for the [136]
(Gastrointestinal treatment against toxoplasmic
tract and brain) encephalitis caused due to Toxoplasma
Gondi. The treatment by atovaquone
shows poor oral bioavailability. Thus,
development of atovaquone
nanocrystals using SDS leads to
improved oral bioavailability and brain
uptake.
Diabet Glibencamide Combinationtec Oral Nanocrystals of poorly soluble [77]
es hnology antidiabetic glibencamide were
transferred to fast dissolving tablets
which retained dissolution properties of
the drug nanocrystals. Study is of
potential clinical relevance.
Hypert Carvedilol Nano Intra-nasal An alternative route of administrating [45]
ension precipitation carvedilol so as to bypass heavy first
aided with ultra- pass metabolism was explored. An in-
sonication situ gelling spray loaded with
nanocrystals was administered intra
nasally to significantly improve the
absolute bioavailability in comparison
to oral route. Nanocrystals sub served
to increase nasal permeability of drug.
Telmisartan Antisolvent Oral 10 fold increase in bioavailability was [164]
precipitation observed
technique
Candesartan Media milling Oral Results suggested that enhanced [165]
cilexetil dissolution velocity and saturation
solubility of nanocrystals extends the
bioavailability of drug
Olmesartan Media milling Oral Nanocrystals seem to overcome the [166]
medoxomil poor oral bioavailability of drug.
1,3- Wet-milled Oral/subcuta Subcutaneous route was found to [167]
dicyclohexylurea neous suitable for administration of
nanocrystals rather than oral.
Antips Ziprasidone Wet milling Oral Nanocrystals improved the fasted state [168]
ychotic bioavailability in beagle dogs when
- compare to commercially available
bipolar capsules.
mania
8. Conclusion
Taking into account potential advantages and reduced side effects, nanocrystals are
considered as suitable candidates for in-vivo delivery of poorly soluble drugs. It is a unique
approach to solve bioavailability related issues. Industrially feasible manufacturing
techniques have served to bring nanocrystals from researcher’s bench to patient’s bed at an
accelerated rate. Nanocrystals are versatile pharmaceuticals which can be delivered through
almost all route of administration such as oral, parenteral, dermal, ocular etc. They are adept
to convenient sterilization techniques, a key driver for development of parenteral
formulations. Incorporation of appropriate stabilizers followed by drying (freeze/spray) can
ensure long term stability of nanocrystals. Dried nanocrystals are suitable for pharmaceutical
processing like granulation which might usher a new era of second generation nanocrystals.
Nanocrystals decorated with functionalized ligands _ENREF_1have generated a new ray of
hope by being able to target various organs with higher affinity. Furthermore, nanocrystals
might be an opportunity to produce generic versions of presently available costly drugs. In
conclusion, engineered nanocrystal technology has huge potential to deliver pre-existing or
newly developed notorious (poorly aqueous soluble) drugs in a more acceptable and effective
dosage form with high commercial applicability.
Acknowledgement
CSIR-CDRI Communication No. 8640
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Engineered Nanocrystal Technology: In-Vivo Fate, Targeting and Applications in Drug Delivery

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Journal of Controlled Release.

2014, 183, 51-66

Engineered Nanocrystal Technology: in-vivo fate,

Pharmaceutics Division, CSIR-Central Drug Research Institute, Lucknow, UP, India

Keywords: Nanocrystals, Poorly Soluble Drugs, In-Vivo Fate, Bioavailability, Stability,

combination approaches and chemical synthesis. Additionally, during the developmental

Table 1.Marketed products formulated using drug nanocrystal technology in healthcare

4. Ensuring long term stability: drying

5. Fate of nanocrystals in biological environment

Fig. 4 (B) Diagrammatic representation of clathrin-mediated, non-clathrin (caveolae) mediated endocytosis of

Table 2 Drug delivery routes explored by nanocrystals

Cyclosporine HPH 962 nm Autoimmune disease [74]

comparison to non-ligated paclitaxel nanocrystals, ligated nanocrystals showed enhanced in-

Table 3 Various disease states targeted by nanocrystals

technology lead to development of a

[51] B. Rabinow, J. Kipp, P. Papadopoulos, J. Wong, J. Glosson, J. Gass, C.-S. Sun, T.

[104] S. Britland, W. Finter, H. Chrystyn, D. Eagland, M.E. Abdelrahim, Droplet

[121] X. Miao, C. Sun, T. Jiang, L. Zheng, T. Wang, S. Wang, Investigation of nanosized

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