International Journal of Pharmaceutics 586 (2020) 119555

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International Journal of Pharmaceutics

journal homepage: www.elsevier.com/locate/ijpharm

Review

Formulation aspects of intravenous nanosuspensions T

Dipeekakumari Patel, Sandeep S. Zode, Arvind K. Bansal
⁎

National Institute of Pharmaceutical Education and Research (NIPER), Sector 67, S.A.S. Nagar, Punjab 160062, India

ARTICLE INFO ABSTRACT

Keywords: Intravenous (IV) route is preferred for rapid onset of action, avoiding first pass metabolism and achieving site
Intravenous delivery specific delivery. Development of IV formulations for poorly water soluble drugs poses significant challenges.
Nanocrystalline suspensions Formulation approaches like salt formation, co-solvents, surfactants and inclusion complexation using cyclo-
Scale-up dextrins are used for solubilisation. However, these approaches are not applicable universally and have lim-
Tissue targeting
itations in extent of solubilisation, hypersensitivity, toxicity and application to only specific type of molecules. IV
Regulatory
nanosuspension have been attracting attention as a viable strategy for development of IV formulations of poorly
water-soluble drugs. Nanosuspension consists of nanocrystals of poorly water soluble drug suspended in aqueous
media and stabilized using minimal concentration of stabilizers. Recent years have witnessed their potential in
formulations for toxicological studies and clinical trials. However various challenges are associated with the
translational development of IV nanosuspensions. Therefore, the objective of the current review is to provide a
holistic view of formulation development and desired properties of IV nanosuspensions. It will also focus on
advancements in characterization tools, manufacturing techniques and post-production processing. Challenges
associated with translational development and regulatory aspects of IV nanosuspension will be addressed.
Additionally, their role in preclinical evaluation and special applications like targeting will also be discussed
with the help of case studies. The applications of IV nanosuspensions shall expand as their applications move
from preclinical phase to commercialization.

1. Introduction drugs, co-solvents, surfactants and inclusion complexes like cyclodex-

New chemical entities (NCE) coming into pipeline are poorly water
soluble due to the emerging role of computational and combinatorial
chemistry (Kipp, 2004). It has been observed that 90% of these new
chemical entities belong to Biopharmaceutics Classification System
(BCS) class II (70%) or class IV (20%). Classification of BCS class II as
per the developability classification system (DCS) follows two sub-
classes: dissolution rate limited class IIa, termed as ‘brick-dust’ mole-
cules, and solubility limited class IIb, the so-called ‘grease-ball’ mole-
cules (Butler and Dressman, 2010). Brick-dust molecules are
characterized by high melting point of > 200 °C and moderate lipo-
philicity (log P < 2). They display poor solubility in water as well as
lipids (eg. itraconazole). Grease-ball molecules have log P of > 3 and
low melting points eg. danazol (log P = 4.53), tamoxifen (log
P = 5.26) (Fagerberg et al., 2010) and show solubility in at least some
lipids (Bergström et al., 2016). For grease-ball molecules, a lipidic
system or an intravenous emulsion can be adopted, provided the drug
has low melting point (Wong et al., 2008).
Formulation strategies to solubilize the brick-dust molecules for an
intravenous (IV) formulation include pH adjustment, using salt form of
trins (Wong et al., 2008). Use of salt form of drug is only applicable to
ionizable drugs and there is risk of in-vivo precipitation due to change in
pH. (Bracq et al., 2008). Hemotoxicity is associated with use of some
co-solvents such as ethanol and polyethylene glycol while higher
amounts of surfactants (like Cremophor) can lead to hypersensitivity
reactions like pseudo-anaphylactoid characterized by pruritus, er-
ythema, rash or generalized urticaria (ten Tije et al., 2003). Cyclodex-
trins used in higher concentrations increases the viscosity of solutions
which is unfavourable in case of IV formulation and can cause ne-
phrotoxicity on prolonged use. They are also not preferred when the
dose of drug is high, as the molar ratio of the cyclodextrins and the drug
is generally 1:1 or 2:1 (Yalkowsky, 1999). IV nanosuspension can be a
viable formulation option for brick-dust molecules having high dose
and grease-ball molecules having high melting point.
Nanosuspension consists of nanocrystals of poorly water soluble
drug suspended in aqueous media and stabilized using low concentra-
tion of stabilizers (Patravale et al., 2004). They have particle size in the
nano scale range (below 1 µm) and thus have potential advantage over
coarse suspension. The small particle size shows significantly rapid
dissolution. Clinically the major benefits of a nanosuspension

⁎
Corresponding author.
E-mail address: akbansal@niper.ac.in (A.K. Bansal).

https://doi.org/10.1016/j.ijpharm.2020.119555
Received 14 March 2020; Received in revised form 23 May 2020; Accepted 14 June 2020
Available online 17 June 2020
0378-5173/ © 2020 Elsevier B.V. All rights reserved.
D. Patel, et al.
Fig. 1. Various technologies for generations of nanocrystals (Sheokand et al., 2018).
formulation include (1) high drug loading that facilitates low volume of
injection, (2) low toxicity due to absence of solubilizing agents, (3)
possible alteration of the pharmacokinetics (PK) of the drug with
modified drug release formulations and (4) targeted delivery of drug by
either active or passive targeting. IV nanosuspension have been at-
tracting lot of attention because of aforementioned potential benefits
(Pace et al., 1999; Rabinow, 2004). Successful formulations have been
reported for antineoplastic agents (Merisko-Liversidge et al., 1996),
anaesthetic agents (Boedeker et al., 1994), antifungals (Rabinow et al.,
2007) and agents for malignant hyperthermia (Karan et al., 1996).
IV nanosuspension are a popular formulation approach during de-
velopment of drug molecules especially when the safety of the NCE
needs to be assessed in experimental animals at multi-fold times of its
intended human dose. It is possible to eliminate the toxic adverse ef-
fects of co-solvents and administer significantly larger doses of drug
(Kesisoglou and Mitra, 2012). Recent years have witnessed their ap-
plications in toxicological and clinical formulations (Frank and Boeck,
2016). Therefore, the drug molecules once abandoned due to solubility
and toxicity issues can now be explored for product development. Al-
though literature is available on nanosuspension in general, very few
reports are available on IV nanocrystalline suspension which can be of
two types i.e. ‘ready to use’ and ‘dry powder nanosuspension for re-
constitution’. This review will focus on formulation development, de-
sired properties, analytical aspects and challenges associated with
translational development of IV nanocrystalline suspension. Ad-
ditionally, their role in preclinical evaluation, special applications like
targeting and regulatory aspects will be addressed.
2. Manufacturing techniques
Nanosuspension can be prepared using three approaches. These can
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International Journal of Pharmaceutics 586 (2020) 119555
be categorized into bottom-up, top-down and combination approaches.
Bottom-up technique involves self-assembling of particles to form na-
noparticles and includes dissolving the drug in an organic solvent fol-
lowed by adding an antisolvent to precipitate the drug in presence of a
stabilizer. Organic solvents and antisolvents are miscible with each
other. Commonly used solvents include ethanol, dimethylsulfoxide,
methanol, dichloromethane and acetone, while water is the most
commonly used antisolvent. Bottom-up approaches include super-
critical fluid processes, sonocrystallization, liquid jet crystallization,
emulsion solvent evaporation and spray drying.(Ahire et al., 2018; Suri
et al., 2016) More recently, a novel bottom-up technique to produce
nanocrystals was developed by controlled crystallization using freeze-
drying.(De Waard et al., 2008).
In top-down technique, larger particles are reduced to nanoparticles
by size reduction techniques. This mainly includes the wet media mil-
ling (WMM) technique, high pressure homogenization (HPH) and mi-
crofluidization technique. In WMM, the coarse drug particles are
charged in a dispersion medium containing milling beads and stabilizer
(s). Particle size reduction is mainly due to mechanical attrition(Gao
et al., 2008a). Contamination of the nanocrystalline suspension can be
due to abrasion of milling glass beads or zirconium beads or parts of
milling chamber. This contamination limits the use of WMM method for
an IV product. However, this challenge was overcome by introduction
of highly crosslinked polystyrene beads as milling media (Kesisoglou
et al., 2007). This media undergoes elastic deformation, thus reducing
formation of cracks and abrasions. The commercial technology Nano-
Crystal® uses PolyMill media which comprises of polystyrene beads.
This leads to nanosuspension with high product purity which can be
used parenterally. Invega Sustenna, a nanocrystal based product, was
generated using WMM based NanoCrystal® technology.
HPH is an alternative technique for the production of nanocrystals
D. Patel, et al.
by top-down approach. In this technique, drug suspension passes at a
high speed through the narrow orifice of the homogenizer. Particle size
reduction is brought about by cavitation, high shear forces and the
collision of the particles against each other (Müller et al., 2011). An-
other technique is microfluidization technique where two fluid streams
collide at very high pressure resulting in generation of nanocrystals. It is
based on the principle of jet-stream homogenization (Shegokar and
Müller, 2010). Combination techniques simply uses both top-down and
bottom-up technologies to generate nanosuspension. Fig. 1 outlines the
various methods that are used in the preparation of nanosuspension
along with their principles, advantages and disadvantages. For detailed
explanation of manufacturing techniques, readers can refer the reviews
by Patravale et. al. (Patravale et al., 2004), Lakshmi et. al. (Lakshmi and
Kumar, 2010), Salazar et. al. (Salazar et al., 2014).
3. Development of IV nanosuspensions:
3.1. Desired formulation properties
3.1.1. Particle size
Particle size of the nanocrystals is a crucial factor for its safety and
in vivo fate. Drug nanocrystals will come across the venules (lumen
diameter of ~ 20 µm) and capillaries (lumen diameter of ~ 6 µm),
following IV administration (Wong et al., 2008). Particles > 7 µm and
larger agglomerates can cause pulmonary embolism and may risk the
patient safety. Therefore, the particle size in a nanocrystalline suspen-
sion should be below 5 µm. It was reported that etoposide-bovine serum
albumin nanosuspension when injected IV (particle sizes of
100–300 nm) was taken up by the tumor cells by means of enhanced
permeation and retention effect (Xiong et al., 2008). Size of the crys-
talline particles can influence the pharmacokinetic behaviour of the
formulation. For example, animal studies conducted with itraconazole
nanosuspension indicated size dependent pharmacokinetic perfor-
mance which was most distinct for larger crystals (i.e.,
those ≥ 340 nm). Size of approximately 100–200 nm is preferred for
rapid dissolution. The pharmacokinetics of such nanosuspension is ex-
pected to be similar to that of an IV solution. When prolonged dis-
solution is required, the mean particle diameter must be in the rela-
tively greater nanometer range, e.g. 800–1000 nm. This prolonged
effect helps in targeting of nanosuspension to the monocyte phagocytic
system (MPS) cells by avoiding complete dissolution of the particles
before they reach the macrophages (Müller et al., 2001).
3.1.2. Sterility
Sterility is one of the most important criteria for any IV formulation.
Sterilization of injectables is achieved either through aseptic proces-
sing, terminal sterilization using autoclaving or gamma radiation (for
dry powders). However, nanosuspension offer a unique challenge as
these methods may deleteriously affect the formulation and its stability.
For example, Shegokar et al. investigated the effect of radiation and
moist heat sterilization on the lyophilized nanosuspension. They con-
cluded that the mean particle size increased with the escalation of ra-
diation dose and with moist heat sterilization (Shegokar and Singh,
2012). Therefore, we need to devise product-specific techniques for
sterilization. One way is to sterilize the API and the excipients sepa-
rately and mix them under aseptic conditions. The raw material drug
can be obtained as a sterile grade API or it can be sterilized by gamma
radiation (Reid, 1995) or by dry heat (Moller and Jensen, 1970) if the
drug is stable to either of the methods. An aqueous solution containing
excipients can be separately sterilized by conventional means and then
mixed with the API under aseptic condition. Another strategy can be
sterile filtration of aqueous nanosuspension prepared using sterile API.
An example of sterile filtration for nanosuspension is the NanoCrsytalTM
for X-ray contrast agent iodipamide. 0.2 µm Supor® 200 DCF™ filter was
used and 100% of Pseudomonas diminuta were retained through it
(Zheng and Bosch, 1997). Radiation sterilization is unacceptable due to
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International Journal of Pharmaceutics 586 (2020) 119555
free radical formation by ionizing radiation. Therefore, it is re-
commended to down process the formulation to a dry powder and then
sterilize it using gamma radiation (Reid, 1995). A radiation dose of
25 kGy is recommended for sterilization of nanosuspension (Shegokar
and Singh, 2012). Sterilization method best suited for the formulation
must be chosen on the basis of formulation components.
3.1.3. Redispersibility and reconstitution time
Sterile powders for reconstitution as nanosuspension can be formed
after downstream processing of the nanosuspension formulation either
by lyophilization or other technique. These powders are supplied with a
suitable sterile diluent (usually water for injection) for reconstitution as
nanosuspension. It is, therefore, essential that the nanocrystals redis-
perse easily with gentle agitation and the nanocrystals maintain their
integrity after reconstitution. USP mentions specific tests of uniformity
of dosage units and water content for sterile powders for suspension
which can be taken into consideration for nanosuspension as well.
3.1.4. Osmolality
The determination of the osmolar concentration of plasma is called
osmolality and it is proportional to the number of particles per kilogram
of solvent. It is usually expressed as mOsmol/kg. As per USP, the os-
molality of blood should range between 285 and 310 mOsmol/kg.
Therefore, the average physiological osmolality is approximately 297.5
mOsmol/kg (Roethlisberger et al., 2017). It has been reported that
hypertonic solutions with an osmolality > 600 mOsmol/kg can cause
shrivelling up of the erythrocytes and cause pain. While hypotonic in-
jection solutions with an osmolality of < 150 mOsmol/kg may cause
haemolysis and pain at the injection site. The limits for osmolality have
to be interpreted with caution because there are several factors like
selection of vein for injection, injection volume and duration that can
cause irritation (Roethlisberger et al., 2017). There are products with
significant hyperosmolality and the tolerance values differs with infu-
sion and small volume injection. Therefore, for drug products intended
for intravenous use, the recommended upper osmolality limit should be
controlled generally under 1000 mOsmol/kg (Wang, 2015).
3.1.5. pH
A formulation with an extreme pH can possibly cause inflammatory
reactions like vascular irritation or pain. Therefore, a target pH range of
3.5–9.0 is recommended for small volume injections to avoid such
complications (Roethlisberger et al., 2017). Buffering agents are used to
adjust the pH will be discussed in latter section. The physiological local
reaction cascade on IV injection depends on a number of factors like
injection volume, injection rate, blood flow, viscosity, cosolvents and
active agents. Therefore, a direct correlation between pain or irritation
and pH cannot be made unless all such factors are kept constant. In case
of a marginal pH, a slow infusion rate (for example, 5-minute push
instead of a quick bolus injection) might help to minimize the risk of
vascular irritation and vein damage. Better tolerance in this case can be
attributed to the slow infusion time and to the increased dilution of
drug by the flow of blood at the injection site (Roethlisberger et al.,
2017). IV drug products with a wide range of pH values (2.55–11.15)
are available in the market as they overcome the drug solubility and
stability issues. This shows that one can optimize the pH of the IV na-
nosuspension in the above mentioned range based on the formulation
development requirement. However, for drugs having pH dependent
solubility, pH should be kept in a narrow range to prevent Ostwald-
ripening.
3.1.6. Endotoxins and pyrogenicity
Endotoxins are lipopolysaccharides found in the outer membrane of
gram negative bacteria and their presence even in nanogram quantities
can elicit immunological response. The endotoxin limit for parenteral
formulation is defined on the basis of dose and is given by the equation
K/M, where K is the threshold human pyrogenic dose of endotoxin per
D. Patel, et al. International Journal of Pharmaceutics 586 (2020) 119555

kg of body weight, and M is the maximum recommended human dose Table 1

per kg of body weight per hour. The endotoxin limit for API used in the Stabilizers used in IV formulations and their IID limits.
formulation must be known at the start of formulation. For parenteral Sr. No. Stabilizer IID limit (Maximum potency
drugs, the endotoxin limit is specified in individual monographs and per unit dose)
expressed in units like EU/ml or EU/unit. In absence of any active
1. Poloxamer 188 0.6% w/v
procedure of subsequently removing the endotoxins, it is important to
2. Polyethylene glycol 400 75.58% w/v
limit the total endotoxins by controlling the specifications of input 3. Polysorbate 20 4.8% w/v
materials in the formulation. An in-house specification can be included, 4. Polysorbate 80 69.33% w/v
to meet the general criteria of injectables. USP Chapter 85 describes the 5. Povidone K12 10% w/v
Bacterial Endotoxins Test based on the use of Limulus Amoebocyte 6. Soybean lecithin 21.3% w/v
7. D-α-Tocopheryl polyethylene glycol 225 mg/L
Lysate (LAL) reagent to detect bacterial endotoxins in the parenteral
1000 succinate (TPGS)
formulation (Chapter85, 2007). This chapter includes the photometric 8. Albumin 2% w/v
techniques (chromogenic and turbidimetric tests) and gel-clot techni-
ques using LAL reagent. Gel-clot technique is more commonly used and
the reaction endpoint is determined by comparison with a reference stabilizers to the particle surface (Verma et al., 2009a). Stabilizer se-
standard. Precautions should be taken with sample preparations and lection can also be done according to their surface tension. The study
dilutions of the test sample and standard sample with LAL reagent. can be performed by contact angle measurement. The stabilizer with
Pyrogenicity testing is an alternative to bacterial endotoxin test. USP lower surface tension and low contact angle acts as a better stabilizer
pyrogen test provides information about requirements for the absence (Chen et al., 2004). The amount and type of stabilizer used in the na-
of pyrogens in a parenteral formulation. The test involves measurement nosuspension formulation has a prominent effect on the physical sta-
of rise in temperature of rabbits following IV administration of drug bility and in-vivo performance of the formulation. Regardless of the
formulation and is limited for products that can be tolerated by test method (bottom-up or top-down or combination) used for the pre-
rabbits in a dose that do not exceed 10 mL per kg injected IV within a paration, the selection of the right type of stabilizer with its optimal
period of not > 10 min. Before the initiation of the test, care should be concentration are essential for stabilizing the smaller size particles. This
taken that all of the excipients used must meet USP limits on endotoxins would help to maintain the shelf life stability of the final product (Van
and all the glasswares are depyrogenated using a validated method. Eerdenbrugh et al., 2009; Wu et al., 2011). However, researchers have
Test nanosuspension formulation must be injected IV into three rabbits, demonstrated the preparation of nanoparticles without using any sta-
the formulation passes the test if none of the animals demonstrate an bilizer. This preparation requires rapid and powerful mixing devices
individual temperature rise of 0.5 °C or more to its corresponding (Wang et al., 2007; Xu et al., 2012).
control temperature for 3 h. A retest procedure has to be followed if the
test fails. The pyrogen test can be used for product release.
3.2.2. Organic solvents
Bottom up process and combination approaches used for generation
3.2. Formulation components
of nanosuspension utilize organic solvents. Organic solvent like
ethanol, isopropyl alcohol, methanol, acetone and N-Methylpyrrolidone
Developing an IV nanosuspension may be challenging as the
can be used for preparing the drug solution (Dong et al., 2009). How-
number of excipients approved for parenteral route are limited in
ever, residual organic solvents may raise toxicity concerns and increase
numbers. The formulation may comprise of stabilizers, buffering
regulatory burden. According to ICH guidelines, class lll solvents can be
agents, tonicity adjusting agents and organic solvents.
used in the preparation of nanocrystals as these solvents are regarded as
less toxic. Class II solvents can also be used if their residual solvent
3.2.1. Stabilizers
remains within the permissible limits. Class I are considered toxic and
Stabilizers are used to wet the drug particles and to prevent
cannot be used in IV formulations. Table 2 consists of the ICH class II
Ostwald’s ripening (Müller et al., 1998). Absence of the stabilizers can
and III organic solvents along with their permissible daily exposure
induce formation of aggregates, due to high surface energy of drug
(PDE) and concentration limits.
nanocrystals. They should be selected based on their interaction with
active pharmaceutical ingredient (API) and their stabilization potential.
Stabilizers can be either ionic surfactants, known to exhibit electrostatic 3.2.3. Buffers
repulsion or polymers which provide steric hindrance and thereby A physiological pH (~7.4) should be the target for IV formulations
prevent particle aggregation. A combination of surfactants and poly- as extreme pH (< pH 3 or > pH 10.5) may cause irritation upon ad-
mers can be used to provide better stabilization. Stabilizers should be ministration due to local cell damage and necrosis (Roethlisberger
selected on the basis of FDA’s Inactive Ingredient Database (IID) for IV et al., 2017). pH of the formulation needs to be controlled to avoid drug
route. Some of the stabilizers that have been explored for IV formula- degradation during processing, storage and reconstitution therefore, it
tions include Pluronic F-68, Polysorbate 20, Lecithin, Polysorbate 80, becomes important to evaluate buffering agents. Selection of the buf-
Pluronic F-108 and vitamin E polyethylene glycol succinate (TPGS) (Li fering agent depends on the pH stability of the API. However, the
et al., 2011). List of FDA approved stabilizers along with their IID limits
for IV route are given in table 1. The ratio of drug and stabilizer may Table 2
vary from 1:20 to 20:1 and must be investigated depending on the ICH class solvents with their PDE concentration limits.
physicochemical properties of API (George and Ghosh, 2013). Further, Solvent Class of PDE (mg/ Concentration limit
surfactant concentration can be decided based on its critical micelle solvent day) (ppm)
concentration (CMC). Care should be taken not to cross the CMC of the
Acetonitrile 2 4.1 410
surfactants, as above CMC micelle formation takes place and surface 1,4- Dioxane 2 3.8 380
adsorption on particles is reduced. Moreover, this leads to increased Methanol 2 30 3000
particle size as micelle formation increases the solubility of API thereby N-Methylpyrrolidone 2 5.3 530
enhancing particle growth by Ostwald ripening (Sinha et al., 2013). Acetone 3 50 5000
Dimethyl sulfoxide 3 50 5000
Toxicity of the surfactants above CMC is also a concern for IV for-
Ethanol 3 50 5000
mulations. Isopropyl alcohol 3 50 5000
Atomic force microscopy (AFM) can be used to study the affinity of

4
D. Patel, et al.
Table 3
List of buffering agents used in IV formulations.
Buffering agents pKa pH range
Maleic acid 1.9, 6.2 2–3
Tartaric acid 2.9, 4.2 2.5–4
Lactic acid 3.8 3–4.5
Citric acid 3.1, 4.8, 6.4 3–7
Acetic acid 4.75 4–6
Sodium bicarbonate 6.3, 10.3 4–9
Sodium phosphate 2.2, 7.2, 12.4 6–8
Sodium hydroxide 13.8 12
Glycine 2.34, 9.6 2.2–3.6; 8.8–10.6
Histidine 1.82, 9.17 5.5–7.4
interaction of the buffer with the solvent system and the drug should be
examined as they are likely to affect surface charge or may interact with
the surfactants/ drug, thus changing their properties. The following
table 3 comprises of the FDA approved buffering agents that can be
commonly used in IV formulations.
3.2.4. Tonicity adjusting agents
Isotonicity is another key requirement, particularly if the product is
intended for large volume infusion, as hypotonic solutions cause hae-
molysis. Hypertonic solutions may be tolerated if administered slowly
in small volumes (Shi et al., 2009). Non-ionic excipients like mannitol
and dextrose have been effectively applied without affecting the PSD
for adjusting the tonicity (Jacobs et al., 2000; Sigfridsson et al., 2007).
Moreover, they provide additional advantage of acting as matrix
forming agent in the downstream processing of the nanosuspension
through spray-drying or freeze drying (discussed in detail in subsequent
section). Salts are usually avoided to adjust the tonicity of the nano-
crystalline suspension as they may destabilize the formulation by al-
tering the electric charge of the nanoparticles.
4. Dry powder nanosuspension for reconstitution
Post-production processing, also known as downstream processing,
of nanosuspension is crucial when the drug is extremely susceptible to
chemical degradation. It may also be essential when the stabilizer
cannot effectively stabilize the formulation for a longer period of time
and the particle size increases as a result of Ostwald ripening. Other
challenges associated with nanosuspension are physical instabilities
like sedimentation, creaming and aggregation. Therefore, it is necessary
to preserve the original size of nanocrystals during their downstream
processing. Techniques such as freeze drying, spray drying, and spray-
freeze drying are reported for the downstream processing of parenteral
nanosuspension.
4.1. Freeze drying
Lyophilization or freeze drying is commonly used technique for the
solidification of nanosuspension. It comprises of freezing, primary
drying and secondary drying steps which leads to the final moisture
content of the formulation below 1%. This dry powder can then be
reconstituted immediately before use to generate nanosuspension and
can be administered intravenously. Cryoprotectants are added into the
formulations to protect the actives from freezing damage. Optimization
of the cryoprotectant type and its concentration is important as it af-
fects the final product quality. Shegokar et.al. investigated dextrose,
lactose, trehalose, mannitol, sorbitol, polyvinyl pyrrolidone, fructose,
sucrose, glucose and reported that their protective effect is concentra-
tion dependant with lower concentrations yielding a sticky product.
Hence it was suggested that the 6–8% concentration of the sugars
should be used to act as an effective protectant. The effect of ster-
ilization process should also be assessed. During the process of
5
International Journal of Pharmaceutics 586 (2020) 119555
lyophilization, particle size of the nanocrystals may increase. In a study,
the authors reported that the particle size was not maintained after
lyophilization. However, the final size obtained was still below 5 µm
which suggested that it could be administered intravenously (Shegokar
and Singh, 2012). Similar results of particle size increments were ob-
tained when doxorubicin loaded chitosan nanoparticles were lyophi-
lized with mannitol and sucrose as cryoprotectant (Dave et al., 2012).
In one of the studies reported, it was observed that after reconstituting
the lyophilized product, the mean size of Ascorbyl palmitate nanosus-
pensions without cryoprotectant (particle size~540 nm) was sig-
nificantly higher compared with the system containing trehalose as
cryoprotectant (1–10% w/w) (particle size of ~360 nm)
(Teeranachaideekul et al., 2008). Therefore, it can be concluded that
the type and amount of cryoprotectant added to the formulation has a
pronounced effect on particle size of the freeze-dried product (Shegokar
and Singh, 2011).
pH shifts in the formulation during lyophilization may adversely
affect the product thereby making it unsuitable for intravenous ad-
ministration. Buffering species present in the formulation may crystal-
lize during the process of lyophilization leading to a drastic shift in pH,
resulting in degradation of the active component. Sodium and po-
tassium phosphate salts crystallize during cooling and in frozen solution
which leads to a decrease in pH of about 4 units and therefore they are
not used in lyophilization (Gómez et al., 2001). Shalaev et al. studied
succinate, citrate and tartrate buffers for their crystallization behaviour
and its effect on pH of the formulation. It was found that citrate buffer
was better in comparison to succinate and tartrate as it remained
amorphous, with pH shift being minimal, while the others crystallized
during lyophilization (Shalaev et al., 2002). It is advisable to place the
buffering agent in the reconstitution fluid if it interacts with the active
component or hinders the lyophilization process.
4.2. Spray drying
Spray drying is routinely used for the conversion of liquid for-
mulation into dry solid powder. In this process, nanosuspension is
atomised to fine droplets in a preheated chamber which are then eva-
porated to form dry powder. Heat and mass transfer occur between air
and the particles which might lead to slight stickiness depending upon
the formulation composition. This stickiness or aggregation can be
circumvented by adding adsorbents such as lactose, mannitol, mal-
todextrose, etc.
The nature of adsorbents with their concentration and spray drying
conditions affects the characteristic of final product. Critical process
parameters for this technique are inlet temperature, feed rate and as-
piration rate. Shegokar et al. studied the effect of different adsorbents
during spray drying. Sugars such as mannitol, lactose and maltodextrin
were chosen as the adsorbents at 2–8% w/w concentration. Spray
drying with maltodextrin and mannitol yielded spherical particles while
lactose produced irregular shaped particles. The pH of the reconstituted
powder was around 7.01–7.11. Lower concentrations of 2% w/w of the
adsorbents resulted in sticky powder. Powder with good flowability was
attained for lactose at higher concentrations of 4–8% during spray
drying (Shegokar and Singh, 2012). In another study, researchers re-
ported that sugars with lower glass transition (Tg) temperatures like
dextrose and sucrose resulted in sticky powders. On the other hand,
lactose and mannitol (Tg > 80 °C) yielded flowable powder. Therefore,
it is suggested that Tg is a better predictor for the selection of ad-
sorbents in comparison with melting point for spray drying perfor-
mance (Chaubal and Popescu, 2008).
Aggregation of particles may be caused due to the excess stress
during the process of drying. Another reason causing aggregation of
particles during spray drying include force of air, velocity, and contact
time between particles (Gianfrancesco et al., 2008). Chaubal et al. in-
vestigated effect of spray drying on itraconazole nanosuspension. They
reported that the mean particle size of spray dried particles was
D. Patel, et al.
10–20% higher than the mean of the original suspension. It was ob-
served that polymeric surfactants and sugars were insufficient to sta-
bilize the nanoparticles during the spray drying process and ad-
ditionally charged surfactant was needed to inhibit irreversible
aggregation (Chaubal and Popescu, 2008). Hence process optimization
is necessary in order to generate redispersed nanosuspension for in-
travenous administration.
4.3. Spray freeze drying
Spray freeze drying (SFD) technique is a combination of two pro-
cesses mentioned above. This technique involves three stages: droplet
generation, freezing and sublimation drying. Droplets are generated by
passing the nanosuspension through a nozzle and they are immediately
frozen in liquid nitrogen. These frozen droplets are now subjected to
lyophilization and water is removed by sublimation drying (Niwa and
Danjo, 2013). SFD is particularly used for thermosensitive products like
recombinant proteins and vaccines. This technique has been explored
for injectables and inhalable products (Wanning et al., 2015). It is a
promising approach for the downstream processing as it offers smaller
particle size when compared to the spray dried product. However, there
are no examples of drying IV nanosuspension using SFD.
4.4. Electrospraying
Electrospraying, also called electrohydrodynamic atomization
(EHDA), allows generation of submicron droplets with a narrow size
distribution. It works on electromechanical and hydrodynamic forces
that function in combination with each other. Nanosuspension is passed
through an emitter (glass or metal capillary) to which high voltage is
applied. Under the influence of electric field, the surface tension of li-
quid causes the liquid jet to break into droplets. These droplets, when
coupled with drying gas, will evaporate to form dry solid particles.
Voltage applied, flow rate and distance between tip to collector are
critical parameters of this process (Chan and Kwok, 2011). Thakkar
et al. made a comparative evaluation between electrospraying and
freeze drying as solidification techniques for erlotinib nanosuspension.
On the basis of various parameters like amount of drug, particle size,
morphology, solid state form of drug, surface area, pore volume, size
after redispersion and drug release, it was reported that lyophilized
powder was superior over electrospray dried powder. However, this
technique was investigated for preparing a nano spray dried powder for
orodispersible tablets (Thakkar et al., 2018). It would be interesting to
explore this technique further for injectables.
5. Characterization of nanosuspension
5.1. Zeta potential
Zeta potential (particle charge) provides an indication about the
physical stability of nanosuspension formulation. Zeta potential can be
determined by Zetasizer and a value of at least ± 30 mV is essential for
an electrostatically stabilized nanosuspension (Müller and Hildebrand,
1996). Indomethacine nanosuspension, stabilized with sodium lauryl
sulfate (electrostatic stabilizer), exhibited higher zeta potential values
ranging from −84 mV to −90 mV whereas lower zeta potential values
were found in the range of −17 mV to −20 mV when HPMC (steric
stabilizer) was used (Verma et al., 2011). In the case of collective sta-
bilization (by electrostatic and steric), a minimum zeta potential value
of ± 20 mV is desirable. In one of the studies, authors investigated
nanosuspensions that were stabilized through a combination of an
electrostatic stabilizer, soya lecithin and a steric stabilizer, tyloxapol.
The zeta potential of budesonide nanosuspension which combination of
stabilizers was found to be −41.1 mV. (Jacobs and Müller, 2002).
Values ranging from −5 to + 5 mV point towards faster aggregation
6
International Journal of Pharmaceutics 586 (2020) 119555
and hence indicates short-term stability (Li et al., 2018). However, this
may not be the essential requirement for stability. Nanosuspensions are
reported to be stable without having zeta potential in the above men-
tioned range.(Verma et al., 2009b) This can be attributed to inherent
differences in the density and effect of gravity between the nanocrys-
talline dispersions and other nanoparticulate systems.
5.2. Particle size and particle size distribution (PSD)
Mean particle size and PSD are important parameters as they control
dissolution velocity, saturation solubility, physical stability and in-vivo
behaviour of nanosuspension. Photon correlation spectroscopy (PCS)
can be used to determine the mean particle diameter and the range of
particle size distribution (polydispersity index, PI) of nanosuspension.
The PI is significant as it dictates the physical stability of nano-for-
mulations and should be low enough for long-term stability of nano-
suspension. A PI value of < 0.3 indicates fairly narrow size distribution.
Although PCS is a versatile and rapid technique, but it only covers the
range of 0.02–0.3 µm (Knapp et al., 1996). Hence, laser diffractrometry
(LD) analysis should be performed additionally to PCS in order to detect
the drug microparticles that might be present in the formulation. LD
analysis becomes critical for IV nanosuspension as it determines a vo-
lume size distribution and can be used to measure particles in the range
of 0.05–80 µm. In some instruments particle sizes up to 2000 µm can be
measured (Keck and Müller, 2008). This is important for IV nanosus-
pension as size > 6 µm can lead to capillary blockage or emboli for-
mation. Another technique that can be employed for determination of
particle size is the Coulter counter technique. The Coulter counter
provides the absolute number of particles per volume unit for the dif-
ferent size classes in the range of 0.4 µm – 1600 µm (Müller and Peters,
1998). Hence, it is a more appropriate and efficient technique than LD
analysis for measuring the microparticulate drug crystals present in
nanosuspension. Both PCS and LD require proper dilution either with
deionized water or stabilizer solution before the analysis to prevent
multiple scattering, thus modifying the nature of nanosuspension to a
certain degree (Li et al., 2015).
Visual characterization techniques like scanning electron micro-
scopy (SEM) and transmission electron microscopy (TEM) can be used
as supportive technique for the identification of primary drug nano-
crystals (Bilgili et al., 2016). With combined use of SEM/ TEM imaging
and particle sizing, the aggregation nature of nanosuspension can be
assessed qualitatively and quantitatively (Li et al., 2018). Due to the use
of high vacuum in these techniques, water evaporation takes place
which obstructs the electron beam and interfere with the image re-
solution.
Other techniques for particle size determination are wide angle X-
ray scattering (WAXS), small-angle X-ray scattering (SAXS) and X-ray
diffraction (XRD). These are indirect methods and need dry powder
sample for analysis, but they offer more reliable information from the
statistical viewpoint. XRD can be used for determining the particle size
from the widening of the XRD reflections by means of the Scherrer
equation, Williamson- Hall plot and Rietveld method. The Scherrer
equation is given by (Scherrer, 1918)
K
D=
cos
where D is the particle size, K is a constant which depends on
crystallite size (close to unity), λ is the wavelength of the radiation, β is
the full width on a 2θ scale at half maximum intensity and θ is the
Bragg’s diffraction angle. In this method, average crystallite size has
been calculated using Gaussian fit by fitting the XRD pattern. According
to Williamson Hall method, (Williamson and Hall, 1953), the individual
contributions to the reflection broadening can be expressed as
D. Patel, et al.
cos = K + [4 sin ]
D
where 4ε sinθ is the effect of strain on the crystallites. This indirect
method of analysis is preferred when higher angle reflections are weak
and difficult to analyze. The expression used in Rietveld method is
expressed as (Young, 1993)
IG
FWHM 2 = (U + DST 2)(tan2 ) + V (tan ) + W +
cos 2
where U, V and W are the usual peak shape parameters; IG is a
measure of the isotropic size effect and DST is the coefficient related to
strain. Particle size of nanoparticles can also be done using surface area
analysis from BET which is a relatively simple technique to calculate
the particle size. However, its limitation is that it is based on the hy-
pothesis of spherical shape of particles for calculation which is not
applicable to the various types of nanoparticles (Suri et al., 2016). More
recently, solid-state NMR spectroscopy (ssNMR) has been explored for
the estimation of particle size in the nanoformulation. Munson et al.
have investigated the correlation between particle size and ssNMR
proton spin–lattice relaxation (1H T1) times. They observed that smaller
particles of dicumarol usually had shorter relaxation times than larger
ones. This technique can be used to determine particle size of APIs even
in presence of excipients and such evaluation of particle size distribu-
tion of the API could be helpful during the process of development.
However, authors propose that there may be other factors that can be
attributed to the change in relaxation time which needs to be explored
to employ ssNMR as a tool for particle size evaluation (Dempah et al.,
2017). Also on-line monitoring of size of particle is now possible with
the help of near-infrared (near-IR) spectroscopy. This technique offers
real-time data for process monitoring with an accuracy of 2.4 nm close
to the endpoint of particle production (Higgins et al., 2003). In con-
clusion, the particle size of nanoparticles needs to be confirmed using
complementary tools to ensure the particle size in the given formula-
tion.
5.3. Particle morphology
Microscopy is the only technique where the distinct particles can be
visually detected, thus providing information about the morphology
besides particle size. Optical microscopy cannot be used in case of
particles which are shorter than the wavelength of visible light. Hence,
electron microscopy technique must be used for particle size in the
nanometer range. Both SEM/ TEM techniques provide images with
highest resolution and can reveal the finest details of the structure
(Bilgili and Afolabi, 2012; Borchert et al., 2005). The sample must be
completely dry before SEM/ TEM analysis and so both SEM and TEM
require proper sample preparation prior to analysis. The first step is
drying the sample which can be done by either freeze drying or freeze
fracture. In case of SEM analysis, the dry sample is made conductive by
coating the surface with gold, palladium, platinum or osmium. For TEM
analysis, a coating of osmium tetraoxide is applied onto the dry sample
to provide sufficient contrast (Müller-Goymann, 2004). However, water
loss may lead to changes in the microstructure of the sample and also
drying alters the existing aggregation state of drug nanosuspension (Li
et al., 2018). Apart from this, rheological characterization of nanosus-
pension could support the assessment of the aggregation state (Azad
et al., 2015). Atomic force microscopy works on the principle of scan-
ning probe microscopy and also reflects the surface topography of
sample (Verma et al., 2009a). Sample preparation for AFM is simple
than SEM/ TEM and the samples do not have to be conductive. The
nanocrystalline particles are to be dispersed or adhered on a substrate
with the help of an adhesive in order to characterize them. Most
commonly used substrates are mica, glass cover slips, highly oriented
pyrolytic graphite (HOPG), atomically flat gold and silicon oxide wafers
and poly-L-Lysine is the commonly used adhesive (Starostina and West,
7
International Journal of Pharmaceutics 586 (2020) 119555
2006).
5.4. Solid state characterization
Process parameters used during generation of nanocrystals may
cause changes in solid form of crystalline particles. Therefore, the final
solid form of drug in the formulation needs to be confirmed as it may
affect stability and its performance after IV administration. It was ob-
served that during the production of nanosuspension, application of
high pressures led to amorphization of API, e.g. the drug RMKP22 was
found in amorphous state in the nanosuspension (Grau and Müller,
1998). Differential scanning calorimetry (DSC) and XRD analysis are
most commonly used to identify the solid form of drug nanocrystals
(Liu et al., 2018). Percent crystallinity can be determined through XRD
by plotting the calibration curve of different fractions of physical
mixtures containing crystalline and amorphous drug. It is imperative to
note that sample preparation is essential to determine the solid form of
API in the final formulation. Downstream processing of the nanosus-
pension involves inclusion of additional excipients which may interfere
with the characterization process. Thermal events of DSC may overlap
with that of the excipients used or excipient-drug interaction might lead
to polymorphic transition. Hence, it is advisable to carry out solid state
characterization of the API alone. This can be implemented by drying
the nanosuspension formulation at ambient conditions. Critical para-
meters during sample preparation are drying temperature and drying
time. Care should be taken that drying temperature or the duration of
the process do not affect the solid form generated during the manu-
facturing process. Additionally, electron microscopy can be used to
characterize the crystalline drug from the amorphous. Crystalline drug
appears bright against black polarized background in the birefringence
mode in contrast to amorphous that appears dark. In conjugation with
DSC, hot stage microscopy (HSM) can help in identifying events asso-
ciated with various thermal events aids differentiation of DSC en-
dotherms for polymorphic transitions.
5.5. In vivo performance
Determination of an in vivo performance of the formulation is an
essential part of the study as it helps in the establishment of an in vitro\
in vivo correlation (IVIVC). In vivo performance is currently evaluated in
costlier, time-consuming rodent models. Utilization of the chick chor-
ioallantoic membrane (CAM) model is a rapid, accessible and low-cost
alternative approach (Leong et al., 2010). CAM is highly vascularized,
mimicking the diverging/converging vasculature of mammalian organs
such as the liver or spleen and is known to trap nanoparticles. In one
study, CAM imaging revealed dramatically different circulation beha-
viours of colloidally stable cationic particles with identical size, shape
and zeta potential, differing only in charge distribution (Townson et al.,
2013). Nanoparticles with patchy charge were immediately seques-
tered, whereas uniformly charged nanoparticles remained in circula-
tion. This observation was later verified within a rodent model via
SPECT imaging (Dogra et al., 2018). Importantly, the CAM model can
also be utilized for nanoparticle–tumour interaction studies (Cho et al.,
2011; Durfee et al., 2016). While rodent models remain indispensable
for new investigational drugs, the CAM model assertively assists as an
inexpensive, efficient intermediary system which can help qualify na-
nosystems for subsequent mammalian testing. This will also reduce the
number of mammalian animals utilized and help bridge the in vitro to in
vivo gap (Leong et al., 2019). The in vivo behaviour of the drug is in-
fluenced by its surface hydrophobicity and interactions with plasma
proteins. The composition of protein adsorption pattern obtained after
the IV injection of nanocrystals is considered as a crucial factor for
organ distribution (Blunk et al., 1993; Blunk et al., 1996; Lück et al.,
1998). Therefore, appropriate techniques must be employed so that the
protein interactions and surface properties can be evaluated. Surface
hydrophobicity can be determined by hydrophobic interaction
D. Patel, et al.
chromatography (Wallis and Müller, 1993) whereas 2-D PAGE (Blunk
et al., 1993) can be utilized for the measurement of protein adsorption
both qualitatively and quantitatively after IV injection of drug nano-
suspensions.
6. Scale-up
Drug nanocrystals are incorporated in the formulation development
decision trees of several pharmaceutical companies as they are amen-
able to scalability. Scalability is achieved by the sameness condition,
the similarity among laboratory, pilot and the production. Majority of
the drug products available in the market have been prepared using
WMM and HPH due to the ease of scalability. In case of WMM, process
parameters and material attributes like the amount of the drug, amount
of milling pearls used, milling speed, milling time and temperature
must be controlled to obtain particles in the desired size range. WMM is
preferred due to the availability of suitable equipment like agitated ball
mills where the drug is milled for about 30–120 min to obtain nano-
crystals of desired size. They can operate in batch mode or continuous
mode (recirculation mode). For large scale operation it is often used in
recirculation mode to obtain the desired particle size. These agitated
mills have media separators like filter cartridge or separating gap
system that allows it to hold the milling media back in the milling
chamber while the nanosuspension is circulating. The suspension is
pumped back into the milling chamber with a certain velocity and
hence within a short residence time in the chamber, the particles are
exposed to high energy input thereby reducing its particle size (Kwade,
1999). Planetary ball mills with modified sample holders are available
for small-scale production of nanosuspension. Alternatively, agitated
ball mills are used for drug quantities starting from 10 mg (Merisko-
Liversidge and Liversidge, 2011). Using these mills, it is now possible to
produce nanosuspensions during the early discovery phase of the for-
mulation development or to perform stabilizer screening studies with a
minimal API consumption. WMM can be viewed as a scalable approach
with the availability of appropriate equipment for small scale to com-
mercial production batch.
HPH is another technique that is commonly used to produce na-
nocrystals. High pressure homogenizers are commonly available in the
pharmaceutical industry and hence can be utilized in the commercial
production of nanosuspension. HPH is unlikely to generate impurities
compared to WMM as a consequence of abrasion and wearing of the
equipment. A comparative study revealed that a typical nanosuspension
after 20 cycles at 1500 bar contained < 1 ppm iron (Krause et al.,
2000). However, in piston-gap homogenizers, wearing and abrasion can
arise when very hard material is processed. This happens because the
tip of the homogenization valve consists of a comparatively small sur-
face in comparison to the volume of suspension moving across it. They
become worn out when stainless steel parts are used thereby affecting
the process efficiency. Therefore, modern homogenizers have valves
fitted with ceramic tips, providing additional protection in harsh con-
ditions making it more suitable for parenteral use (Innings et al., 2011).
In HPH, the coarser suspension is passed several times (i.e. homo-
genization cycles) to achieve the desired particle size. In order to avoid
clogging of the narrow homogenization gap, the applied pressure is
increased from 10% to 100%. Production pressure usually spans be-
tween 1000 and 2000 bar wherein the opening gap is only of a few
micrometer (Möschwitzer, 2013). There are reports that describe de-
creased microorganism load and enzyme inactivation as a result of HPH
process (Dumay et al., 2013). Today, homogenizers are available from
mL-scale to commercial production scale, making HPH a scalable pro-
cess (Keck and Müller, 2006). In one of the studies, a comparative
evaluation was made between product produced at lab scale and pilot
scale using WMM and HPH. The authors concluded that the particle size
of the nanosuspension was dependent on the velocity of pumping,
agitator rotation speed and the number of milling cycles. Particle size
reduction by HPH was observed to be affected by the applied pressure
8
International Journal of Pharmaceutics 586 (2020) 119555
and number of homogenization cycles (Al Shaal et al., 2010). It is
usually recommended to start with micronized suspension for both HPH
and WMM processes since clogging of the equipment can occur when
the process is steered with coarse suspension. All combination tech-
nologies mentioned earlier can perform better than standard WMM and
HPH, however, any pre-treatment step would increase the complexity
of the entire process and can add to the overall cost (Möschwitzer,
2013).
7. Regulatory aspects
The US Food and Drug Administration (FDA) regulates nano-
technology products within the existing statutory legal standards ap-
plicable to each type of product. Technical assessments are made by the
agency on the basis of the product and science-based policy, thus al-
lowing tailored-made approaches that reveal the characteristics of
particular products (e.g., nanocrystals), besides incorporating evolving
technology and scientific understanding within the field. In 2014, FDA
released a guidance document for industry that describes the agency’s
thinking on considering whether FDA- regulated products involve the
application of nanotechnology. This guidance document considers two
points in evaluating if the product involves application of nano-
technology: (1) a material or end product is generated to have at least
one external or an internal dimension or structure in the nanometer
range (approximately 1 to 100 nm), (2) an end product is engineered to
unveil physical or chemical properties or biological effects that are
attributable to its dimensions up to 1000 nm.
Generic products containing nanocrystal APIs can be submitted to
the FDA for approval through 505 (b) (2) route that allows the appli-
cant to rely on the studies or data previously available in the original
application or published in the literature. For example, Ryanodex®
(dantrolene sodium) is the nanocrystalline product which is a re-
formulated product from Dantrium Intravenous, a lyophilized for-
mulation (micronized) for injection. One of the reports revealed that
submissions for nanocrystals comprises of approximately 30% of all the
applications for drug products containing nanomaterials (Chen et al.,
2017). Nanocrystalline suspension for oral or intravenous administra-
tion are generated through the top-down and bottom-up techniques
mentioned earlier. Hence, their critical quality attributes (CQAs) gov-
erning the quality remain the same.
A nanocrystalline drug product specification includes the CQAs
encompassing PSD, zeta potential, particle shape, polymorphism, drug
content and drug release.(Chen et al., 2017; Peltonen, 2018) Several
process variables like critical material attributes (CMAs) and critical
process parameters (CPPs) must be controlled with the aim of achieving
the desired CQAs. Examples of CQAs for drug nanocrystals and how
they are affected by various process variables like CPPs and CMAs are
captured in Table 4. Various quality considerations from chemistry,
manufacturing and controls (CMC) perspective also includes control of
drug substance and drug product and their stability. It is important to
include crystallinity as well as PSD in the drug substance specifications
for nanocrystals. The agency proposes to subject the products (invol-
ving application of nanotechnology) to premarket review. In case the
products are not subjected to premarket review process, the FDA urges
the industry to consult the agency in the initial stage of product de-
velopment.
The European Nanomedicine Characterisation Laboratory (EUNCL)
and the REFINE consortium effort, funded by EC-H2020, are aimed at
developing a regulatory science framework for nanomedicine. EUNCL/
REFINE, jointly with the National Cancer Institute’s Nanotechnology
Characterization Lab (NCI-NCL), would bridge the gap between pub-
lication and translation by identifying common pitfalls in nanomedicine
development. It would also define critical quality attributes for pre-
clinical assessment thus enabling product development (Caputo et al.,
2019; Crist et al., 2013; Gioria et al., 2018; Swierczewska et al., 2018).
D. Patel, et al. International Journal of Pharmaceutics 586 (2020) 119555

Table 4
Example for risk assessment matrix for drug nanocrystals analyzing the impact of CMAs and CPPs on CQAs (Peltonen, 2018).
Risk assessment matrix
CQAs CMAs CPPs
Stabilizer type Stabilizer concentration Drug amount Milling time Milling speed Bead size

Particle size Medium Medium Medium High High High

PDI Medium High Medium High High High
Zeta potential High High Medium Medium Medium Medium
Drug content Low Low Low Low Low Low
Drug release Medium Medium Medium High High High

8. Applications the mixture, there is reduced contamination and interruption amongst

the runs. This process has been developed by ResodynTM corporation
8.1. Role in preclinical formulations with their ResonantAcoustic® Mixing method and various commercial
acoustic mixers are available.
Preclinical formulations are an integral part of drug discovery and
development. Their development follows both the stages of drug de-
8.2. Targeting of nanosuspension
velopment i.e. at discovery stage (lead discovery) and at preclinical
stages (preclinical lead) (Li and Zhao, 2007). These early formulations
Nanocrystals have recently emerged as a formulation strategy to
help to optimize in vivo exposure, predict initial dosing for human
target drugs at specific site of action for a better therapeutic effect.
clinical trials and reduce toxicological side effects. General tox-
Nanoparticulate systems have revealed great potential in delivery of
icological (tox) studies are carried out to assess the adverse effects of
drugs to the targeted organs of the body (Chen et al., 2018). Similarly,
investigational compounds in vivo. Tox formulations present significant
nanocrystalline suspension can be used for targeting of drugs due to
challenges to a formulation scientist as these formulations demand high
their surface potential and their in vivo behaviour.
concentrations and they are used for a long time period (Li and Zhao,
Following IV injection, drug nanocrystals can extravasate to the
2007). For example, in dose escalation studies, it is necessary that the
tumors from the leaky endothelium via the enhanced permeation and
formulation delivers numerous folds greater in vivo exposure than the
retention effect (EPR). This phenomenon is known as passive targeting
effective dose and also the preparation is dosed incessantly for definite
which is mainly a result the submicron size of nanocrystalline particles
period of time (for example, 2 weeks). It is important to note that the
(Maeda, 2015). Nanocrystals may behave analogous to the drug solu-
vehicle is anticipated not to provoke any adverse effect. Toxicological
tion after IV administration due to rapid dissolution profile achieved in
studies are critical for a lead compound on its way to progress. Dis-
in vivo sink conditions (Mouton et al., 2006). If their dissolution is not
covery leads usually are in limited quantity (for example, < 20–30 mg)
immediate then, they are taken up by macrophages predominantly in
and hence the early formulation development is a challenge if the drug
the liver (Kupffer cells) and spleen, later diffusing slowing across the
compound has poor solubility.
concentration gradient (Rabinow, 2004). This IV depot action of the
Preclinical leads require detailed efficacy/activity, pharmacokinetic
formulation offers a pharmacokinetic profile with prolonged t1/2 and
and toxicological profile. Requirement of a formulation selection is
significantly reduced Cmax. This behaviour of nanocrystals is beneficial
more specific here. Nanosuspensions can serve as an immediate solu-
for some classes of drug, for whom toxicity is intervened by peak
tion for the preclinical formulations as they provide high drug con-
plasma values, nonetheless the efficacy is determined by AUC as ob-
centrations and are devoid of a large number of excipients (Kesisoglou
served in the event of antifungals (Andes, 2003). In a clinical trial
and Mitra, 2012). Nanosuspension for early formulations can be de-
(Donnelly et al., 2001), the pharmacokinetic parameters of nanocrys-
veloped using the NanoMill® (< 50 mL) or high pressure homogenizers
talline suspension of itraconazole were compared to the commercial
(provided they handle small volumes efficiently). In one of the case
Sporanox solution. The nanosuspension with particle size of about
studies, it was reported that a neutral compound having 2 µg/mL as its
200–300 nm employed a larger volume of distribution (1,677 ± 827 l
solubility and a log P of 2.5 was to be tested in monkeys. The drug
versus 796 ± 185 l) and a slow clearance (3.35 ± 1.8 l/h versus
compound was not adequately lipidic to be incorporated in a lipidic
22.9 ± 5.7 l/h) to provide a longer t1/2 (346 ± 225 h terminal versus
system and solubilizing agents like Captisol®, vitamin E TPGS, HPβCD
35.4 ± 29.4 h mean and 30 h terminal) and larger area under the
proved to be futile to solubilize the drug. Hence, a nanosuspension was
plasma concentration curve for the initial 24 h, AUC24,
developed using the microfluidization technique which generated a
(51,558 ± 10,635 (g•h/l versus 30,605 ± 8,961 (g•h/l) (Willems
particle size (D50) of 40 nm. When such nanosuspension formulation
et al., 2001). The study presented above confirms the mononuclear
was administered to monkeys, it attained 130% of the exposure of the
phagocyte system (MPS) depot behaviour, resulting in delivery for
standard suspension (Boersen et al., 2013). There are reports available
itraconazole nanosuspension for a prolonged period of time. In another
in the literature where nanosuspension have been successfully devel-
finding, it was reported that itraconazole nanosuspension having an
oped for preclinical studies. For example, a poorly water soluble com-
average particle size of 268.1 ± 6.5 nm showed similar dissolution
pound, 1,3- Diclyclohexylurea (DCU), which lowers blood pressure was
profile to that of marketed itraconazole injections. However, it dis-
developed as nanosuspension for infusion and IV bolus dosing
played different pharmacokinectic properties such as decreased initial
(Wahlstrom et al., 2007). In another study, nanosuspension for in-
drug concentration, increased mean residence time (MRT) and plasma
travenous administration in preclinical studies of a poorly aqueous
half-life, and increased concentration in the liver, spleen, and lung in
soluble compound BI XX was developed using ball mill (Frank and
comparison to commercial itraconazole injections. Therefore, nano-
Boeck, 2016).
suspension could change the in vivo distribution of itraconazole also
A novel drug sparing technology employing acoustic mixing is re-
helping in passive targeting to MPS (Yuan et al., 2019)
ported to identify nanosuspension formulations for widespread com-
In one of the studies, it was reported that clofazimine nanosuspen-
pounds with enhanced efficiencies (Leung et al., 2014). This technique
sion (particle size 385 nm) administered intravenously demonstrated
applies high intensity acoustic energy coupled with low frequency to
targeting to the reticuloendothelial system (RES), successfully targeting
the mixer with low shear to generate nanosuspension. Since the mixer
the drug to major host cells in mycobacterial infections i.e. tissue
agitates the whole container and has no contact with the components of
macrophages. Since clofazimine has low solubility in water (0.3 g/l at

9
D. Patel, et al.
pH 7.8), these nanocrystals did not dissolve immediately and resulted in
the sequestration of nanocrystals in the RES (Peters et al., 2000). Ganta
et.al. reported that asulacrine (ASL) nanosuspension with average size,
133 ± 20 nm, was rapidly cleared from the plasma and showed re-
markably increased concentrations in tissues like the liver, lungs and
kidney. The authors have rationalized that this was due to the uptake of
the nanocrystals by the MPS which cleared them from systemic circu-
lation. Therefore, ASL nanosuspension had a distinctly larger AUC0-∞ in
kidney and liver but not in heart (Ganta et al., 2009). Gao et al. have
studied particle size effect on the tissue distribution and pharmacoki-
netics of two oridonin NCs with distinctly different sizes
(103.3 ± 1.5 nm and 897.2 ± 14.2 nm) following IV administration
in rabbits. Complete dissolution was observed in vitro for the smaller
and larger NCs in 10 min and 2 h, respectively. In vivo, the smaller NCs
performed in the same way as drug solution, while the larger NCs ac-
cumulated to a greater degree in the liver, lungs and spleen. The au-
thors proposed that the small crystals dissolved completely in short
span of time in vivo thus behaving like solution whereas the larger NCs
remained insoluble and were subjected to MPS uptake (Gao et al.,
2008b). This is in line with the studies discussed earlier. In another
study, Zhang et al. demonstrated a superior antitumor efficacy for un-
coated camptothecin NCs having a particle size of 240 nm in a MCF- 7
tumor xenograft mouse model in comparison to the camptothecin salt
solution made of propylene glycol and saline. This was ascribed to the
EPR effect and higher hydrolysis resistance of NCs. They additionally
supported their observations with biodistribution data presenting
greater deposition of camptothecin in the tumor and reduced drug
hydrolysis (Zhang et al., 2011).
It is evident from the aforementioned studies that altering the par-
ticle size plays an important role in targeting of nanocrystals. Smaller
crystals target tumour cells by virtue of their EPR effect and larger
crystals are up taken by MPS predominantly in the spleen and liver.
Apart from this, targeting ligands can be used for surface mod-
ification of drug nanocrystals. Surface modification will also help to
prolong the in vivo dissolution of nanocrystals. Sharma et al. in-
vestigated hyaluronic acid (HA) anchored paclitaxel (PTX) nanocrystals
(HA-PTX/NC) for its chemotherapeutic efficacy. It was observed that
HA-PTX/NC markedly prolonged the systemic circulation of PTX and
increased the AUC0-∞ by 8.4 times in comparison with marketed for-
mulation of PTX i.e. TaxolTM. The study also revealed that HA-PTX/NC
exhibited reduced lung metastatis, greater antitumor efficacy, and less
toxicity in LA-7 tumor bearing rat model in comparison to TaxolTM
(Sharma et al., 2016). In another study, PTX-NC, surface modification
with HA and apo-transferrin (Tf), was assessed for their inhibition of
cell growth. In vitro results presented higher cell permeability in con-
trast to the unchanged PTX-NC. Also in MCF-7 cells the surface mod-
ification of PTX-NCs led to 60% cell growth inhibition, but the effect
was inferior than unmodified PTX-NC and pure PTX in normal cell line
(Sohn et al., 2017). Noh et al. prepared herceptin (HCT) functionalised
PTX-NCs and studied their effect on HER-2 positive breast cancer cells.
The cell uptake studies revealed that surface modified nanocrystals
exhibited higher binding affinity and greater cell-specific uptake to
HER-2 positive breast cancer cell lines than PTX-NCs (Noh et al., 2016).
Shubar et al. investigated the role of apolipoprotein E (apo E) coat on
the surface of atovaquone nanocrystals (ANCs). It was observed that
incubation of apo E coated ANCs with brain endothelial cells resulted in
the accumulation of nanoparticles on the surface but not in their uptake
into the brain cells (Shubar et al., 2009). Shegokar et al. developed
nevirapine nanosuspension with surface modification by dextran,
serum albumin, and polyethylene glycol to improve its targeting action.
In vivo tissue distribution studies showed an increased NCs accumula-
tion in various organs like brain, liver and spleen thereby prolonging
their residence at target site as compared to pure drug (Shegokar and
Singh, 2011). Pandey et al. prepared docetaxel (DTX) nanocrystals and
stabilized them with a novel chondroitin sulphate A (CSA) and poly-
ethylene glycol congujate. This stabilizer provided multiple advantages
10
International Journal of Pharmaceutics 586 (2020) 119555
of pegylation, stabilization and CD44 receptor targeting. It was ob-
served that in MDA-MB-231 cells, DTX-CSA NCs exhibited enhanced
cellular uptake, deeper penetration and exhibited higher degree of cy-
totoxicity. The authors concluded that this was due to the EPR effect
combined with receptor mediated endocytosis. IV administration of
DTX-CSA formulation demonstrated improved pharmacokinetic profile
and significant inhibition in 4T1 induced tumor with reduced toxicity
(Pandey et al., 2018).
8.2.1. Challenges due to shedding of ligands
Stabilizers and targeting ligands are non-covalently linked onto the
surface of nanocrystals and nonspecific interactions like adsorption are
predominant during surface modification. A weak interaction is pro-
vided by physical adsorption and leads to desorption of stabilizer
during the dissolution of nanocrystals. Excessive dilutions faced during
in vitro or in vivo experiments may result in shedding of the stabilizer or
targeting ligands. For example, Deng et al. have demonstrated with the
help of increased crystal size that poloxamer 407 (Pluronic® F127)
detached from paclitaxel nanocrystals on dilution or mild heating
(Deng et al., 2010). Therefore, shedding of ligands/ stabilizers is a
critical obstacle when the aim is to offer targeting to a particular site.
Chemically altering the stabilizer to provide more sites for interac-
tion with nanocrystals can be one of the approaches to reduce shedding.
Another approach includes crosslinking the stabilizer around drug na-
nocrystals thereby reducing shedding via physical entrapment and
physisorption. For instance, Fuhrmann et al. crosslinked block copo-
lymer directly on the exterior of PTX NCs to form polymeric nanocages.
It was reported that these nanocages-NCs provided steric barrier to
particle–particle interaction and prevented aggregation. Size-stability
analysis revealed that the nanocages-NCs had better size-stability when
compared with non-cross-linked coating. It was demonstrated that the
nanocages showed 3 to 4 times less shedding from the NCs surface
which was achieved by tailoring the extent of polymer release from the
nanocage-NCs. Transmission electron microscopic images obtained
after dissolution of NCs were evident that the nanocages remained in-
tact (Fuhrmann et al., 2012). Kim and Lee have reported crosslinking of
chitosan by tripolyphospate on the surface of PTX and naproxen. The
crosslinking significantly modified the release profiles of the NCs.
However, they did not evaluate the size stability and shedding phe-
nomenon after crosslinking (Kim and Lee, 2011).
More recently, the Layer-by-Layer (LbL) assembly of polyelec-
trolytes has been explored as an approach to obtain stabilized coatings
for the encapsulation of drug NCs. In this approach, first step is to an-
chor a layer of small amphiphile molecule and a polymer to the hy-
drophobic drug NC tailed by deposition of many layers of charged
polyelectrolytes sequentially. The drug release is prolonged due to the
thickness of the polymer layers which controls the diffusion of drug out
of the encapsulation. Polomska et al. adopted the LbL approach to coat
PTX NCs with alternating layers of oppositely charged polyelectrolytes
and the top coat with a PEGylated copolymer. It was observed that
dissolution of coated particles was slower than the noncoated NCs and
Abraxane (marketed formulation of PTX). However, the pharmacoki-
netic and biodistribution profiles indicated that the coated NCs were
rapidly removed from the blood stream. The authors hypothesized that
this was due to the shedding of PEGylated coating from the surface of
NCs (Polomska et al., 2017).
In some cases, this shedding of stabilizers may be beneficial. For
instance, Liu et al. studied the shedding phenomenon by preparing PTX
NCs stabilized with D-α-tocopheryl polyethylene gycol 1000 succinate
(TPGS). The rationale of this study was that TPGS acts as a P-gp in-
hibitor which on shedding improved the drug uptake in resistant cells.
This was indeed demonstrated that in NCI/ADR-RES cells, over-
expressing P-gp and PTX resistant, TPGS stabilized formulation pre-
sented a distinctly improved anti-proliferative effect in comparison to
free PTX or poloxamer 407 stabilized PTX formulation (Liu et al.,
2010).
D. Patel, et al. International Journal of Pharmaceutics 586 (2020) 119555

9. Marketed and pipeline nanosuspension products for IV

Marketed

Phase 3
Phase 2
administration

Status
In case of IV delivery, after the approval of a nanoparticulate IV

Eagle Pharmaceuticals
suspension- Abraxane® in 2005, there has been a steady increase in the
drug particulate formulations moving into human clinical trials.

Pharma company
Abraxane® consists of paclitaxel protein-bound particles wherein pa-

Recro Pharma
clitaxel is entrapped in an albumin matrix. However, paclitaxel exists in

Berg, LLC
a non-crystalline i.e. amorphous state in Abraxane® which is different
than the nanocrystal-based nanosuspensions discussed here. Drug na-
nocrystals are one of the most successful formulations that have wit-

technology
nessed short time frame between inventions to market stage. However,
this is true for oral and other routes of administration. Few products

Applied Technology
have now reached clinical phases and only one product is available in

TM
the market for IV administration. This may be due to challenges with

NanoCrystals
the translation development, sterility and long-term stability of these
formulations. Ryanodex® is the only commercial product for IV route. It
is a lyophilized formulation of dantrolene sodium which is recon-

–
stituted with sterile water for injection to form nanosuspension. Apart

Mannitol, polysorbate 80, povidone K12, sodium hydroxide or hydrochloric acid

from this, meloxicam nanocrystals for IV route have reached in the late
phase of clinical trials (phase 3) and ubidecaronone has reached phase
2 clinical trials. Table 5 summarizes the marketed and pipeline nano-
suspension products for IV administration and Table 6 consists of re-
ported nanosuspension for IV administration in the literature.

10. Future perspective

Advancements in formulation approaches and commercially fea-

sible manufacturing processes have aided in bringing nanosuspension
from researcher's lab to patient's bed at an augmented rate. There are a
few nanosuspension based intramuscular (IM) injections already in the
market like Invega Sustenna (Janssen Pharmaceuticals) and
Aripiprazole Lauroxil (Alkermes, Inc.) and Aripiprazole long acting
injection (once a month) by Otsuka Pharmaceuticals used to treat
Schizophrenia. Long acting GSK1265744 (744) and rilpivirine
for pH adjustment.

(TMC278) nanosuspension achieved steady state plasma concentrations

Excipients used

within 3 days which proved its potential use in HIV-1 treatment (Spreen
et al., 2014). This trend is likely to increase in the future as pharma-
ceutical companies are looking at creating nanosuspension based IM
formulations because of their unique advantages of ease of adminis-
–
–
Marketed and pipeline nanocrystalline suspension products for IV administration.

tration and reduction in irritation or pain at the injection site. They are
Acute post-operative pain
Malignant hyperthermia

either depending upon the use of an oily medium or solvent or lipo-

philic prodrugs to obtain extended action. These formulations are
Pancreatic cancer
Therapeutic uses

especially useful in population that are susceptible to noncompliance of

dose e.g. patients with C.N.S. disorders like Schizophrenia. Similarly, in
other immunocompromised patients who have to take large number of
pills daily, it is going to offer a very convenient way of administration
of drugs. IV nanosuspension are pretty common formulation strategy in
the preclinical studies as they allow determination of IV pharmacoki-
netics. However, there are complication associated with the accumu-
lation of drug nanocrystals in the different organs like the RES systems
100 mg/kg (IV
1–2.5 mg/ kg

leading to organ toxicity. Unless these challenges are overcome, com-

mercial products seem to have a difficulty from a regulatory perspec-
infusion
30 mg

tive. At the same time, drugs that are sparingly soluble could be good
Dose

candidates for IV administration because they behave like IV solutions

after administration by virtue of their relative better pharmacokinetics.
Dantrolene sodium

Recent advances in aseptic technologies such as barrier isolation tech-

Ubidecaronone

nology aids in dealing with the technical hurdles of the aseptic pro-
Meloxicam

cessing requirements. Further the development of nanocrystals deco-

Drug

rated with functionalized surface coatings capable of targeting various

organs with high affinity can be viewed as the upcoming stage in the
nanosuspension research.
Trade Name

BPM31510
Ryanodex®
Table 5

N1539

11
 D. Patel, et al.

Table 6
Reported nanosuspension for IV administration.
API Excipients Solvents Manufacturing technique Sterilization Critical parameters Particle size

Itraconazole Poloxamer 188 N-methyl- 2- Microcrystallization followed by Sterilized isolator was used for – 581 ± 18 nm
pyrrolidone HPH microcrytsallization
Omeprazole 1% Poloxamer 188 in 8.4% sodium bicarbonate – DissoCubes – Temperature, Number of 598–603 nm
homogenization cycles, Drug
content
Asulacrine Poloxamer 188 at 1% w/v – Pre homogenization using Ultra- – Temperature 133 ± 20 nm
turrax followed by HPH
Azoxystrobin 0.9% w/w of 1-Dodecanesulfonic acid sodium salt, – Wet media milling – Milling speed and time 238.1 ± 1.5 nm
polyvinylpyrrolidone K30
Camptothecin No additional stabilizers were added DMSO, Sonication with Antisolvent – 200–700 nm
Acetonitrile precipitation method
Curcumin 0.2% w/v Combination of soya lecithin and sodium – HPH – Number of homogenization cycles 132.6 to 360.8

12
deoxycholate and pressure
Clofazimine 0.5% Pluronic F68, 0.6% Phospholipon 90, 0.25% – HPH Aseptic conditions Number of homogenization cycles −385 nm LD (99) value-
Sodium cholic acid, 5.6% mannitol and pressure 2.28 μm
Tarazepide 1% w/w Poloxamer 188, 0.5% w/w Tween 80, Glycerol – HPH Production under laminar air flow Number of homogenization cycles 347–517 nm
as osmotic agent cabinet and pressure
Melarsoprol 1% Poloxamer 188 or 1% Poloxamer 407, 0.5% – HPH – Homogenization pressure, number 324 ± 88 to 663 ± 129 nm
mannitol of cycles
Docetaxel Chondroitin sulphate A-polyethylene glycol conjugate – HPH – Homogenization pressure, number 195.6 ± 12.5 nm
of cycles
Nevirapine 2.8% (w/w) of surfactant solution, Tween 80 (1%), – HPH – Homogenization pressure, number 468.9–520.3 nm
VolpoL4 (0.9%), Plasdone (0.1%), Poloxamer 188 of cycles
(0.5%), PVP (0.3%)
Oridonin 0.5% w/v Poloxamer and Lecithin (molar ratio 3:1), 1% – HPH – Homogenization pressure, number 103.3 ± 1.5 nm and
mannitol as cryoprotectant of cycles 897.2 ± 14.2 nm
Atovaquone 0.3% Tween 80, 0.3% Poloxamer 188 and 0.05% – High pressure homogenization Aseptic processing Homogenization pressure, number 279–286 nm
sodium cholate, glycerol as osmotic agent of cycles
Nimodipine 0.6% (w/v) poloxamer 188, 0.4% (w/v) sodium cholic – High pressure homogenization γ-ray radiation Homogenization pressure, number 300–700 nm
acid and 4.0% (w/v) mannitol of cycles
International Journal of Pharmaceutics 586 (2020) 119555
D. Patel, et al.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
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