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1 s2.0 S0378517320305391 Main
1 s2.0 S0378517320305391 Main
Review
National Institute of Pharmaceutical Education and Research (NIPER), Sector 67, S.A.S. Nagar, Punjab 160062, India
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.
⁎
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. International Journal of Pharmaceutics 586 (2020) 119555
formulation include (1) high drug loading that facilitates low volume of be categorized into bottom-up, top-down and combination approaches.
injection, (2) low toxicity due to absence of solubilizing agents, (3) Bottom-up technique involves self-assembling of particles to form na-
possible alteration of the pharmacokinetics (PK) of the drug with noparticles and includes dissolving the drug in an organic solvent fol-
modified drug release formulations and (4) targeted delivery of drug by lowed by adding an antisolvent to precipitate the drug in presence of a
either active or passive targeting. IV nanosuspension have been at- stabilizer. Organic solvents and antisolvents are miscible with each
tracting lot of attention because of aforementioned potential benefits other. Commonly used solvents include ethanol, dimethylsulfoxide,
(Pace et al., 1999; Rabinow, 2004). Successful formulations have been methanol, dichloromethane and acetone, while water is the most
reported for antineoplastic agents (Merisko-Liversidge et al., 1996), commonly used antisolvent. Bottom-up approaches include super-
anaesthetic agents (Boedeker et al., 1994), antifungals (Rabinow et al., critical fluid processes, sonocrystallization, liquid jet crystallization,
2007) and agents for malignant hyperthermia (Karan et al., 1996). emulsion solvent evaporation and spray drying.(Ahire et al., 2018; Suri
IV nanosuspension are a popular formulation approach during de- et al., 2016) More recently, a novel bottom-up technique to produce
velopment of drug molecules especially when the safety of the NCE nanocrystals was developed by controlled crystallization using freeze-
needs to be assessed in experimental animals at multi-fold times of its drying.(De Waard et al., 2008).
intended human dose. It is possible to eliminate the toxic adverse ef- In top-down technique, larger particles are reduced to nanoparticles
fects of co-solvents and administer significantly larger doses of drug by size reduction techniques. This mainly includes the wet media mil-
(Kesisoglou and Mitra, 2012). Recent years have witnessed their ap- ling (WMM) technique, high pressure homogenization (HPH) and mi-
plications in toxicological and clinical formulations (Frank and Boeck, crofluidization technique. In WMM, the coarse drug particles are
2016). Therefore, the drug molecules once abandoned due to solubility charged in a dispersion medium containing milling beads and stabilizer
and toxicity issues can now be explored for product development. Al- (s). Particle size reduction is mainly due to mechanical attrition(Gao
though literature is available on nanosuspension in general, very few et al., 2008a). Contamination of the nanocrystalline suspension can be
reports are available on IV nanocrystalline suspension which can be of due to abrasion of milling glass beads or zirconium beads or parts of
two types i.e. ‘ready to use’ and ‘dry powder nanosuspension for re- milling chamber. This contamination limits the use of WMM method for
constitution’. This review will focus on formulation development, de- an IV product. However, this challenge was overcome by introduction
sired properties, analytical aspects and challenges associated with of highly crosslinked polystyrene beads as milling media (Kesisoglou
translational development of IV nanocrystalline suspension. Ad- et al., 2007). This media undergoes elastic deformation, thus reducing
ditionally, their role in preclinical evaluation, special applications like formation of cracks and abrasions. The commercial technology Nano-
targeting and regulatory aspects will be addressed. 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
2. Manufacturing techniques generated using WMM based NanoCrystal® technology.
HPH is an alternative technique for the production of nanocrystals
Nanosuspension can be prepared using three approaches. These can
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D. Patel, et al. International Journal of Pharmaceutics 586 (2020) 119555
by top-down approach. In this technique, drug suspension passes at a free radical formation by ionizing radiation. Therefore, it is re-
high speed through the narrow orifice of the homogenizer. Particle size commended to down process the formulation to a dry powder and then
reduction is brought about by cavitation, high shear forces and the sterilize it using gamma radiation (Reid, 1995). A radiation dose of
collision of the particles against each other (Müller et al., 2011). An- 25 kGy is recommended for sterilization of nanosuspension (Shegokar
other technique is microfluidization technique where two fluid streams and Singh, 2012). Sterilization method best suited for the formulation
collide at very high pressure resulting in generation of nanocrystals. It is must be chosen on the basis of formulation components.
based on the principle of jet-stream homogenization (Shegokar and
Müller, 2010). Combination techniques simply uses both top-down and 3.1.3. Redispersibility and reconstitution time
bottom-up technologies to generate nanosuspension. Fig. 1 outlines the Sterile powders for reconstitution as nanosuspension can be formed
various methods that are used in the preparation of nanosuspension after downstream processing of the nanosuspension formulation either
along with their principles, advantages and disadvantages. For detailed by lyophilization or other technique. These powders are supplied with a
explanation of manufacturing techniques, readers can refer the reviews suitable sterile diluent (usually water for injection) for reconstitution as
by Patravale et. al. (Patravale et al., 2004), Lakshmi et. al. (Lakshmi and nanosuspension. It is, therefore, essential that the nanocrystals redis-
Kumar, 2010), Salazar et. al. (Salazar et al., 2014). perse easily with gentle agitation and the nanocrystals maintain their
integrity after reconstitution. USP mentions specific tests of uniformity
3. Development of IV nanosuspensions: of dosage units and water content for sterile powders for suspension
which can be taken into consideration for nanosuspension as well.
3.1. Desired formulation properties
3.1.4. Osmolality
3.1.1. Particle size The determination of the osmolar concentration of plasma is called
Particle size of the nanocrystals is a crucial factor for its safety and osmolality and it is proportional to the number of particles per kilogram
in vivo fate. Drug nanocrystals will come across the venules (lumen of solvent. It is usually expressed as mOsmol/kg. As per USP, the os-
diameter of ~ 20 µm) and capillaries (lumen diameter of ~ 6 µm), molality of blood should range between 285 and 310 mOsmol/kg.
following IV administration (Wong et al., 2008). Particles > 7 µm and Therefore, the average physiological osmolality is approximately 297.5
larger agglomerates can cause pulmonary embolism and may risk the mOsmol/kg (Roethlisberger et al., 2017). It has been reported that
patient safety. Therefore, the particle size in a nanocrystalline suspen- hypertonic solutions with an osmolality > 600 mOsmol/kg can cause
sion should be below 5 µm. It was reported that etoposide-bovine serum shrivelling up of the erythrocytes and cause pain. While hypotonic in-
albumin nanosuspension when injected IV (particle sizes of jection solutions with an osmolality of < 150 mOsmol/kg may cause
100–300 nm) was taken up by the tumor cells by means of enhanced haemolysis and pain at the injection site. The limits for osmolality have
permeation and retention effect (Xiong et al., 2008). Size of the crys- to be interpreted with caution because there are several factors like
talline particles can influence the pharmacokinetic behaviour of the selection of vein for injection, injection volume and duration that can
formulation. For example, animal studies conducted with itraconazole cause irritation (Roethlisberger et al., 2017). There are products with
nanosuspension indicated size dependent pharmacokinetic perfor- significant hyperosmolality and the tolerance values differs with infu-
mance which was most distinct for larger crystals (i.e., sion and small volume injection. Therefore, for drug products intended
those ≥ 340 nm). Size of approximately 100–200 nm is preferred for for intravenous use, the recommended upper osmolality limit should be
rapid dissolution. The pharmacokinetics of such nanosuspension is ex- controlled generally under 1000 mOsmol/kg (Wang, 2015).
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- 3.1.5. pH
tively greater nanometer range, e.g. 800–1000 nm. This prolonged A formulation with an extreme pH can possibly cause inflammatory
effect helps in targeting of nanosuspension to the monocyte phagocytic reactions like vascular irritation or pain. Therefore, a target pH range of
system (MPS) cells by avoiding complete dissolution of the particles 3.5–9.0 is recommended for small volume injections to avoid such
before they reach the macrophages (Müller et al., 2001). complications (Roethlisberger et al., 2017). Buffering agents are used to
adjust the pH will be discussed in latter section. The physiological local
3.1.2. Sterility reaction cascade on IV injection depends on a number of factors like
Sterility is one of the most important criteria for any IV formulation. injection volume, injection rate, blood flow, viscosity, cosolvents and
Sterilization of injectables is achieved either through aseptic proces- active agents. Therefore, a direct correlation between pain or irritation
sing, terminal sterilization using autoclaving or gamma radiation (for and pH cannot be made unless all such factors are kept constant. In case
dry powders). However, nanosuspension offer a unique challenge as of a marginal pH, a slow infusion rate (for example, 5-minute push
these methods may deleteriously affect the formulation and its stability. instead of a quick bolus injection) might help to minimize the risk of
For example, Shegokar et al. investigated the effect of radiation and vascular irritation and vein damage. Better tolerance in this case can be
moist heat sterilization on the lyophilized nanosuspension. They con- attributed to the slow infusion time and to the increased dilution of
cluded that the mean particle size increased with the escalation of ra- drug by the flow of blood at the injection site (Roethlisberger et al.,
diation dose and with moist heat sterilization (Shegokar and Singh, 2017). IV drug products with a wide range of pH values (2.55–11.15)
2012). Therefore, we need to devise product-specific techniques for are available in the market as they overcome the drug solubility and
sterilization. One way is to sterilize the API and the excipients sepa- stability issues. This shows that one can optimize the pH of the IV na-
rately and mix them under aseptic conditions. The raw material drug nosuspension in the above mentioned range based on the formulation
can be obtained as a sterile grade API or it can be sterilized by gamma development requirement. However, for drugs having pH dependent
radiation (Reid, 1995) or by dry heat (Moller and Jensen, 1970) if the solubility, pH should be kept in a narrow range to prevent Ostwald-
drug is stable to either of the methods. An aqueous solution containing ripening.
excipients can be separately sterilized by conventional means and then
mixed with the API under aseptic condition. Another strategy can be 3.1.6. Endotoxins and pyrogenicity
sterile filtration of aqueous nanosuspension prepared using sterile API. Endotoxins are lipopolysaccharides found in the outer membrane of
An example of sterile filtration for nanosuspension is the NanoCrsytalTM gram negative bacteria and their presence even in nanogram quantities
for X-ray contrast agent iodipamide. 0.2 µm Supor® 200 DCF™ filter was can elicit immunological response. The endotoxin limit for parenteral
used and 100% of Pseudomonas diminuta were retained through it formulation is defined on the basis of dose and is given by the equation
(Zheng and Bosch, 1997). Radiation sterilization is unacceptable due to K/M, where K is the threshold human pyrogenic dose of endotoxin per
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D. Patel, et al. International Journal of Pharmaceutics 586 (2020) 119555
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D. Patel, et al. International Journal of Pharmaceutics 586 (2020) 119555
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D. Patel, et al. International Journal of Pharmaceutics 586 (2020) 119555
10–20% higher than the mean of the original suspension. It was ob- and hence indicates short-term stability (Li et al., 2018). However, this
served that polymeric surfactants and sugars were insufficient to sta- may not be the essential requirement for stability. Nanosuspensions are
bilize the nanoparticles during the spray drying process and ad- reported to be stable without having zeta potential in the above men-
ditionally charged surfactant was needed to inhibit irreversible tioned range.(Verma et al., 2009b) This can be attributed to inherent
aggregation (Chaubal and Popescu, 2008). Hence process optimization differences in the density and effect of gravity between the nanocrys-
is necessary in order to generate redispersed nanosuspension for in- talline dispersions and other nanoparticulate systems.
travenous administration.
4.3. Spray freeze drying 5.2. Particle size and particle size distribution (PSD)
Spray freeze drying (SFD) technique is a combination of two pro- Mean particle size and PSD are important parameters as they control
cesses mentioned above. This technique involves three stages: droplet dissolution velocity, saturation solubility, physical stability and in-vivo
generation, freezing and sublimation drying. Droplets are generated by behaviour of nanosuspension. Photon correlation spectroscopy (PCS)
passing the nanosuspension through a nozzle and they are immediately can be used to determine the mean particle diameter and the range of
frozen in liquid nitrogen. These frozen droplets are now subjected to particle size distribution (polydispersity index, PI) of nanosuspension.
lyophilization and water is removed by sublimation drying (Niwa and The PI is significant as it dictates the physical stability of nano-for-
Danjo, 2013). SFD is particularly used for thermosensitive products like mulations and should be low enough for long-term stability of nano-
recombinant proteins and vaccines. This technique has been explored suspension. A PI value of < 0.3 indicates fairly narrow size distribution.
for injectables and inhalable products (Wanning et al., 2015). It is a Although PCS is a versatile and rapid technique, but it only covers the
promising approach for the downstream processing as it offers smaller range of 0.02–0.3 µm (Knapp et al., 1996). Hence, laser diffractrometry
particle size when compared to the spray dried product. However, there (LD) analysis should be performed additionally to PCS in order to detect
are no examples of drying IV nanosuspension using SFD. the drug microparticles that might be present in the formulation. LD
analysis becomes critical for IV nanosuspension as it determines a vo-
4.4. Electrospraying 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
Electrospraying, also called electrohydrodynamic atomization measured (Keck and Müller, 2008). This is important for IV nanosus-
(EHDA), allows generation of submicron droplets with a narrow size pension as size > 6 µm can lead to capillary blockage or emboli for-
distribution. It works on electromechanical and hydrodynamic forces mation. Another technique that can be employed for determination of
that function in combination with each other. Nanosuspension is passed particle size is the Coulter counter technique. The Coulter counter
through an emitter (glass or metal capillary) to which high voltage is provides the absolute number of particles per volume unit for the dif-
applied. Under the influence of electric field, the surface tension of li- ferent size classes in the range of 0.4 µm – 1600 µm (Müller and Peters,
quid causes the liquid jet to break into droplets. These droplets, when 1998). Hence, it is a more appropriate and efficient technique than LD
coupled with drying gas, will evaporate to form dry solid particles. analysis for measuring the microparticulate drug crystals present in
Voltage applied, flow rate and distance between tip to collector are nanosuspension. Both PCS and LD require proper dilution either with
critical parameters of this process (Chan and Kwok, 2011). Thakkar deionized water or stabilizer solution before the analysis to prevent
et al. made a comparative evaluation between electrospraying and multiple scattering, thus modifying the nature of nanosuspension to a
freeze drying as solidification techniques for erlotinib nanosuspension. certain degree (Li et al., 2015).
On the basis of various parameters like amount of drug, particle size, Visual characterization techniques like scanning electron micro-
morphology, solid state form of drug, surface area, pore volume, size scopy (SEM) and transmission electron microscopy (TEM) can be used
after redispersion and drug release, it was reported that lyophilized as supportive technique for the identification of primary drug nano-
powder was superior over electrospray dried powder. However, this crystals (Bilgili et al., 2016). With combined use of SEM/ TEM imaging
technique was investigated for preparing a nano spray dried powder for and particle sizing, the aggregation nature of nanosuspension can be
orodispersible tablets (Thakkar et al., 2018). It would be interesting to assessed qualitatively and quantitatively (Li et al., 2018). Due to the use
explore this technique further for injectables. of high vacuum in these techniques, water evaporation takes place
which obstructs the electron beam and interfere with the image re-
5. Characterization of nanosuspension solution.
Other techniques for particle size determination are wide angle X-
5.1. Zeta potential ray scattering (WAXS), small-angle X-ray scattering (SAXS) and X-ray
diffraction (XRD). These are indirect methods and need dry powder
Zeta potential (particle charge) provides an indication about the sample for analysis, but they offer more reliable information from the
physical stability of nanosuspension formulation. Zeta potential can be statistical viewpoint. XRD can be used for determining the particle size
determined by Zetasizer and a value of at least ± 30 mV is essential for from the widening of the XRD reflections by means of the Scherrer
an electrostatically stabilized nanosuspension (Müller and Hildebrand, equation, Williamson- Hall plot and Rietveld method. The Scherrer
1996). Indomethacine nanosuspension, stabilized with sodium lauryl equation is given by (Scherrer, 1918)
sulfate (electrostatic stabilizer), exhibited higher zeta potential values
ranging from −84 mV to −90 mV whereas lower zeta potential values K
D=
were found in the range of −17 mV to −20 mV when HPMC (steric cos
stabilizer) was used (Verma et al., 2011). In the case of collective sta-
bilization (by electrostatic and steric), a minimum zeta potential value where D is the particle size, K is a constant which depends on
of ± 20 mV is desirable. In one of the studies, authors investigated crystallite size (close to unity), λ is the wavelength of the radiation, β is
nanosuspensions that were stabilized through a combination of an the full width on a 2θ scale at half maximum intensity and θ is the
electrostatic stabilizer, soya lecithin and a steric stabilizer, tyloxapol. Bragg’s diffraction angle. In this method, average crystallite size has
The zeta potential of budesonide nanosuspension which combination of been calculated using Gaussian fit by fitting the XRD pattern. According
stabilizers was found to be −41.1 mV. (Jacobs and Müller, 2002). to Williamson Hall method, (Williamson and Hall, 1953), the individual
Values ranging from −5 to + 5 mV point towards faster aggregation contributions to the reflection broadening can be expressed as
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D. Patel, et al. International Journal of Pharmaceutics 586 (2020) 119555
2006).
cos = K + [4 sin ]
D
5.4. Solid state characterization
where 4ε sinθ is the effect of strain on the crystallites. This indirect
method of analysis is preferred when higher angle reflections are weak Process parameters used during generation of nanocrystals may
and difficult to analyze. The expression used in Rietveld method is cause changes in solid form of crystalline particles. Therefore, the final
expressed as (Young, 1993) solid form of drug in the formulation needs to be confirmed as it may
IG affect stability and its performance after IV administration. It was ob-
FWHM 2 = (U + DST 2)(tan2 ) + V (tan ) + W +
cos 2 served that during the production of nanosuspension, application of
high pressures led to amorphization of API, e.g. the drug RMKP22 was
where U, V and W are the usual peak shape parameters; IG is a found in amorphous state in the nanosuspension (Grau and Müller,
measure of the isotropic size effect and DST is the coefficient related to 1998). Differential scanning calorimetry (DSC) and XRD analysis are
strain. Particle size of nanoparticles can also be done using surface area most commonly used to identify the solid form of drug nanocrystals
analysis from BET which is a relatively simple technique to calculate (Liu et al., 2018). Percent crystallinity can be determined through XRD
the particle size. However, its limitation is that it is based on the hy- by plotting the calibration curve of different fractions of physical
pothesis of spherical shape of particles for calculation which is not mixtures containing crystalline and amorphous drug. It is imperative to
applicable to the various types of nanoparticles (Suri et al., 2016). More note that sample preparation is essential to determine the solid form of
recently, solid-state NMR spectroscopy (ssNMR) has been explored for API in the final formulation. Downstream processing of the nanosus-
the estimation of particle size in the nanoformulation. Munson et al. pension involves inclusion of additional excipients which may interfere
have investigated the correlation between particle size and ssNMR with the characterization process. Thermal events of DSC may overlap
proton spin–lattice relaxation (1H T1) times. They observed that smaller with that of the excipients used or excipient-drug interaction might lead
particles of dicumarol usually had shorter relaxation times than larger to polymorphic transition. Hence, it is advisable to carry out solid state
ones. This technique can be used to determine particle size of APIs even characterization of the API alone. This can be implemented by drying
in presence of excipients and such evaluation of particle size distribu- the nanosuspension formulation at ambient conditions. Critical para-
tion of the API could be helpful during the process of development. meters during sample preparation are drying temperature and drying
However, authors propose that there may be other factors that can be time. Care should be taken that drying temperature or the duration of
attributed to the change in relaxation time which needs to be explored the process do not affect the solid form generated during the manu-
to employ ssNMR as a tool for particle size evaluation (Dempah et al., facturing process. Additionally, electron microscopy can be used to
2017). Also on-line monitoring of size of particle is now possible with characterize the crystalline drug from the amorphous. Crystalline drug
the help of near-infrared (near-IR) spectroscopy. This technique offers appears bright against black polarized background in the birefringence
real-time data for process monitoring with an accuracy of 2.4 nm close mode in contrast to amorphous that appears dark. In conjugation with
to the endpoint of particle production (Higgins et al., 2003). In con- DSC, hot stage microscopy (HSM) can help in identifying events asso-
clusion, the particle size of nanoparticles needs to be confirmed using ciated with various thermal events aids differentiation of DSC en-
complementary tools to ensure the particle size in the given formula- dotherms for polymorphic transitions.
tion.
5.5. In vivo performance
5.3. Particle morphology
Determination of an in vivo performance of the formulation is an
Microscopy is the only technique where the distinct particles can be essential part of the study as it helps in the establishment of an in vitro\
visually detected, thus providing information about the morphology in vivo correlation (IVIVC). In vivo performance is currently evaluated in
besides particle size. Optical microscopy cannot be used in case of costlier, time-consuming rodent models. Utilization of the chick chor-
particles which are shorter than the wavelength of visible light. Hence, ioallantoic membrane (CAM) model is a rapid, accessible and low-cost
electron microscopy technique must be used for particle size in the alternative approach (Leong et al., 2010). CAM is highly vascularized,
nanometer range. Both SEM/ TEM techniques provide images with mimicking the diverging/converging vasculature of mammalian organs
highest resolution and can reveal the finest details of the structure such as the liver or spleen and is known to trap nanoparticles. In one
(Bilgili and Afolabi, 2012; Borchert et al., 2005). The sample must be study, CAM imaging revealed dramatically different circulation beha-
completely dry before SEM/ TEM analysis and so both SEM and TEM viours of colloidally stable cationic particles with identical size, shape
require proper sample preparation prior to analysis. The first step is and zeta potential, differing only in charge distribution (Townson et al.,
drying the sample which can be done by either freeze drying or freeze 2013). Nanoparticles with patchy charge were immediately seques-
fracture. In case of SEM analysis, the dry sample is made conductive by tered, whereas uniformly charged nanoparticles remained in circula-
coating the surface with gold, palladium, platinum or osmium. For TEM tion. This observation was later verified within a rodent model via
analysis, a coating of osmium tetraoxide is applied onto the dry sample SPECT imaging (Dogra et al., 2018). Importantly, the CAM model can
to provide sufficient contrast (Müller-Goymann, 2004). However, water also be utilized for nanoparticle–tumour interaction studies (Cho et al.,
loss may lead to changes in the microstructure of the sample and also 2011; Durfee et al., 2016). While rodent models remain indispensable
drying alters the existing aggregation state of drug nanosuspension (Li for new investigational drugs, the CAM model assertively assists as an
et al., 2018). Apart from this, rheological characterization of nanosus- inexpensive, efficient intermediary system which can help qualify na-
pension could support the assessment of the aggregation state (Azad nosystems for subsequent mammalian testing. This will also reduce the
et al., 2015). Atomic force microscopy works on the principle of scan- number of mammalian animals utilized and help bridge the in vitro to in
ning probe microscopy and also reflects the surface topography of vivo gap (Leong et al., 2019). The in vivo behaviour of the drug is in-
sample (Verma et al., 2009a). Sample preparation for AFM is simple fluenced by its surface hydrophobicity and interactions with plasma
than SEM/ TEM and the samples do not have to be conductive. The proteins. The composition of protein adsorption pattern obtained after
nanocrystalline particles are to be dispersed or adhered on a substrate the IV injection of nanocrystals is considered as a crucial factor for
with the help of an adhesive in order to characterize them. Most organ distribution (Blunk et al., 1993; Blunk et al., 1996; Lück et al.,
commonly used substrates are mica, glass cover slips, highly oriented 1998). Therefore, appropriate techniques must be employed so that the
pyrolytic graphite (HOPG), atomically flat gold and silicon oxide wafers protein interactions and surface properties can be evaluated. Surface
and poly-L-Lysine is the commonly used adhesive (Starostina and West, hydrophobicity can be determined by hydrophobic interaction
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D. Patel, et al. International Journal of Pharmaceutics 586 (2020) 119555
chromatography (Wallis and Müller, 1993) whereas 2-D PAGE (Blunk and number of homogenization cycles (Al Shaal et al., 2010). It is
et al., 1993) can be utilized for the measurement of protein adsorption usually recommended to start with micronized suspension for both HPH
both qualitatively and quantitatively after IV injection of drug nano- and WMM processes since clogging of the equipment can occur when
suspensions. the process is steered with coarse suspension. All combination tech-
nologies mentioned earlier can perform better than standard WMM and
6. Scale-up HPH, however, any pre-treatment step would increase the complexity
of the entire process and can add to the overall cost (Möschwitzer,
Drug nanocrystals are incorporated in the formulation development 2013).
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 7. Regulatory aspects
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 The US Food and Drug Administration (FDA) regulates nano-
parameters and material attributes like the amount of the drug, amount technology products within the existing statutory legal standards ap-
of milling pearls used, milling speed, milling time and temperature plicable to each type of product. Technical assessments are made by the
must be controlled to obtain particles in the desired size range. WMM is agency on the basis of the product and science-based policy, thus al-
preferred due to the availability of suitable equipment like agitated ball lowing tailored-made approaches that reveal the characteristics of
mills where the drug is milled for about 30–120 min to obtain nano- particular products (e.g., nanocrystals), besides incorporating evolving
crystals of desired size. They can operate in batch mode or continuous technology and scientific understanding within the field. In 2014, FDA
mode (recirculation mode). For large scale operation it is often used in released a guidance document for industry that describes the agency’s
recirculation mode to obtain the desired particle size. These agitated thinking on considering whether FDA- regulated products involve the
mills have media separators like filter cartridge or separating gap application of nanotechnology. This guidance document considers two
system that allows it to hold the milling media back in the milling points in evaluating if the product involves application of nano-
chamber while the nanosuspension is circulating. The suspension is technology: (1) a material or end product is generated to have at least
pumped back into the milling chamber with a certain velocity and one external or an internal dimension or structure in the nanometer
hence within a short residence time in the chamber, the particles are range (approximately 1 to 100 nm), (2) an end product is engineered to
exposed to high energy input thereby reducing its particle size (Kwade, unveil physical or chemical properties or biological effects that are
1999). Planetary ball mills with modified sample holders are available attributable to its dimensions up to 1000 nm.
for small-scale production of nanosuspension. Alternatively, agitated Generic products containing nanocrystal APIs can be submitted to
ball mills are used for drug quantities starting from 10 mg (Merisko- the FDA for approval through 505 (b) (2) route that allows the appli-
Liversidge and Liversidge, 2011). Using these mills, it is now possible to cant to rely on the studies or data previously available in the original
produce nanosuspensions during the early discovery phase of the for- application or published in the literature. For example, Ryanodex®
mulation development or to perform stabilizer screening studies with a (dantrolene sodium) is the nanocrystalline product which is a re-
minimal API consumption. WMM can be viewed as a scalable approach formulated product from Dantrium Intravenous, a lyophilized for-
with the availability of appropriate equipment for small scale to com- mulation (micronized) for injection. One of the reports revealed that
mercial production batch. submissions for nanocrystals comprises of approximately 30% of all the
HPH is another technique that is commonly used to produce na- applications for drug products containing nanomaterials (Chen et al.,
nocrystals. High pressure homogenizers are commonly available in the 2017). Nanocrystalline suspension for oral or intravenous administra-
pharmaceutical industry and hence can be utilized in the commercial tion are generated through the top-down and bottom-up techniques
production of nanosuspension. HPH is unlikely to generate impurities mentioned earlier. Hence, their critical quality attributes (CQAs) gov-
compared to WMM as a consequence of abrasion and wearing of the erning the quality remain the same.
equipment. A comparative study revealed that a typical nanosuspension A nanocrystalline drug product specification includes the CQAs
after 20 cycles at 1500 bar contained < 1 ppm iron (Krause et al., encompassing PSD, zeta potential, particle shape, polymorphism, drug
2000). However, in piston-gap homogenizers, wearing and abrasion can content and drug release.(Chen et al., 2017; Peltonen, 2018) Several
arise when very hard material is processed. This happens because the process variables like critical material attributes (CMAs) and critical
tip of the homogenization valve consists of a comparatively small sur- process parameters (CPPs) must be controlled with the aim of achieving
face in comparison to the volume of suspension moving across it. They the desired CQAs. Examples of CQAs for drug nanocrystals and how
become worn out when stainless steel parts are used thereby affecting they are affected by various process variables like CPPs and CMAs are
the process efficiency. Therefore, modern homogenizers have valves captured in Table 4. Various quality considerations from chemistry,
fitted with ceramic tips, providing additional protection in harsh con- manufacturing and controls (CMC) perspective also includes control of
ditions making it more suitable for parenteral use (Innings et al., 2011). drug substance and drug product and their stability. It is important to
In HPH, the coarser suspension is passed several times (i.e. homo- include crystallinity as well as PSD in the drug substance specifications
genization cycles) to achieve the desired particle size. In order to avoid for nanocrystals. The agency proposes to subject the products (invol-
clogging of the narrow homogenization gap, the applied pressure is ving application of nanotechnology) to premarket review. In case the
increased from 10% to 100%. Production pressure usually spans be- products are not subjected to premarket review process, the FDA urges
tween 1000 and 2000 bar wherein the opening gap is only of a few the industry to consult the agency in the initial stage of product de-
micrometer (Möschwitzer, 2013). There are reports that describe de- velopment.
creased microorganism load and enzyme inactivation as a result of HPH The European Nanomedicine Characterisation Laboratory (EUNCL)
process (Dumay et al., 2013). Today, homogenizers are available from and the REFINE consortium effort, funded by EC-H2020, are aimed at
mL-scale to commercial production scale, making HPH a scalable pro- developing a regulatory science framework for nanomedicine. EUNCL/
cess (Keck and Müller, 2006). In one of the studies, a comparative REFINE, jointly with the National Cancer Institute’s Nanotechnology
evaluation was made between product produced at lab scale and pilot Characterization Lab (NCI-NCL), would bridge the gap between pub-
scale using WMM and HPH. The authors concluded that the particle size lication and translation by identifying common pitfalls in nanomedicine
of the nanosuspension was dependent on the velocity of pumping, development. It would also define critical quality attributes for pre-
agitator rotation speed and the number of milling cycles. Particle size clinical assessment thus enabling product development (Caputo et al.,
reduction by HPH was observed to be affected by the applied pressure 2019; Crist et al., 2013; Gioria et al., 2018; Swierczewska et al., 2018).
8
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
9
D. Patel, et al. International Journal of Pharmaceutics 586 (2020) 119555
pH 7.8), these nanocrystals did not dissolve immediately and resulted in of pegylation, stabilization and CD44 receptor targeting. It was ob-
the sequestration of nanocrystals in the RES (Peters et al., 2000). Ganta served that in MDA-MB-231 cells, DTX-CSA NCs exhibited enhanced
et.al. reported that asulacrine (ASL) nanosuspension with average size, cellular uptake, deeper penetration and exhibited higher degree of cy-
133 ± 20 nm, was rapidly cleared from the plasma and showed re- totoxicity. The authors concluded that this was due to the EPR effect
markably increased concentrations in tissues like the liver, lungs and combined with receptor mediated endocytosis. IV administration of
kidney. The authors have rationalized that this was due to the uptake of DTX-CSA formulation demonstrated improved pharmacokinetic profile
the nanocrystals by the MPS which cleared them from systemic circu- and significant inhibition in 4T1 induced tumor with reduced toxicity
lation. Therefore, ASL nanosuspension had a distinctly larger AUC0-∞ in (Pandey et al., 2018).
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- 8.2.1. Challenges due to shedding of ligands
netics of two oridonin NCs with distinctly different sizes Stabilizers and targeting ligands are non-covalently linked onto the
(103.3 ± 1.5 nm and 897.2 ± 14.2 nm) following IV administration surface of nanocrystals and nonspecific interactions like adsorption are
in rabbits. Complete dissolution was observed in vitro for the smaller predominant during surface modification. A weak interaction is pro-
and larger NCs in 10 min and 2 h, respectively. In vivo, the smaller NCs vided by physical adsorption and leads to desorption of stabilizer
performed in the same way as drug solution, while the larger NCs ac- during the dissolution of nanocrystals. Excessive dilutions faced during
cumulated to a greater degree in the liver, lungs and spleen. The au- in vitro or in vivo experiments may result in shedding of the stabilizer or
thors proposed that the small crystals dissolved completely in short targeting ligands. For example, Deng et al. have demonstrated with the
span of time in vivo thus behaving like solution whereas the larger NCs help of increased crystal size that poloxamer 407 (Pluronic® F127)
remained insoluble and were subjected to MPS uptake (Gao et al., detached from paclitaxel nanocrystals on dilution or mild heating
2008b). This is in line with the studies discussed earlier. In another (Deng et al., 2010). Therefore, shedding of ligands/ stabilizers is a
study, Zhang et al. demonstrated a superior antitumor efficacy for un- critical obstacle when the aim is to offer targeting to a particular site.
coated camptothecin NCs having a particle size of 240 nm in a MCF- 7 Chemically altering the stabilizer to provide more sites for interac-
tumor xenograft mouse model in comparison to the camptothecin salt tion with nanocrystals can be one of the approaches to reduce shedding.
solution made of propylene glycol and saline. This was ascribed to the Another approach includes crosslinking the stabilizer around drug na-
EPR effect and higher hydrolysis resistance of NCs. They additionally nocrystals thereby reducing shedding via physical entrapment and
supported their observations with biodistribution data presenting physisorption. For instance, Fuhrmann et al. crosslinked block copo-
greater deposition of camptothecin in the tumor and reduced drug lymer directly on the exterior of PTX NCs to form polymeric nanocages.
hydrolysis (Zhang et al., 2011). It was reported that these nanocages-NCs provided steric barrier to
It is evident from the aforementioned studies that altering the par- particle–particle interaction and prevented aggregation. Size-stability
ticle size plays an important role in targeting of nanocrystals. Smaller analysis revealed that the nanocages-NCs had better size-stability when
crystals target tumour cells by virtue of their EPR effect and larger compared with non-cross-linked coating. It was demonstrated that the
crystals are up taken by MPS predominantly in the spleen and liver. nanocages showed 3 to 4 times less shedding from the NCs surface
Apart from this, targeting ligands can be used for surface mod- which was achieved by tailoring the extent of polymer release from the
ification of drug nanocrystals. Surface modification will also help to nanocage-NCs. Transmission electron microscopic images obtained
prolong the in vivo dissolution of nanocrystals. Sharma et al. in- after dissolution of NCs were evident that the nanocages remained in-
vestigated hyaluronic acid (HA) anchored paclitaxel (PTX) nanocrystals tact (Fuhrmann et al., 2012). Kim and Lee have reported crosslinking of
(HA-PTX/NC) for its chemotherapeutic efficacy. It was observed that chitosan by tripolyphospate on the surface of PTX and naproxen. The
HA-PTX/NC markedly prolonged the systemic circulation of PTX and crosslinking significantly modified the release profiles of the NCs.
increased the AUC0-∞ by 8.4 times in comparison with marketed for- However, they did not evaluate the size stability and shedding phe-
mulation of PTX i.e. TaxolTM. The study also revealed that HA-PTX/NC nomenon after crosslinking (Kim and Lee, 2011).
exhibited reduced lung metastatis, greater antitumor efficacy, and less More recently, the Layer-by-Layer (LbL) assembly of polyelec-
toxicity in LA-7 tumor bearing rat model in comparison to TaxolTM trolytes has been explored as an approach to obtain stabilized coatings
(Sharma et al., 2016). In another study, PTX-NC, surface modification for the encapsulation of drug NCs. In this approach, first step is to an-
with HA and apo-transferrin (Tf), was assessed for their inhibition of chor a layer of small amphiphile molecule and a polymer to the hy-
cell growth. In vitro results presented higher cell permeability in con- drophobic drug NC tailed by deposition of many layers of charged
trast to the unchanged PTX-NC. Also in MCF-7 cells the surface mod- polyelectrolytes sequentially. The drug release is prolonged due to the
ification of PTX-NCs led to 60% cell growth inhibition, but the effect thickness of the polymer layers which controls the diffusion of drug out
was inferior than unmodified PTX-NC and pure PTX in normal cell line of the encapsulation. Polomska et al. adopted the LbL approach to coat
(Sohn et al., 2017). Noh et al. prepared herceptin (HCT) functionalised PTX NCs with alternating layers of oppositely charged polyelectrolytes
PTX-NCs and studied their effect on HER-2 positive breast cancer cells. and the top coat with a PEGylated copolymer. It was observed that
The cell uptake studies revealed that surface modified nanocrystals dissolution of coated particles was slower than the noncoated NCs and
exhibited higher binding affinity and greater cell-specific uptake to Abraxane (marketed formulation of PTX). However, the pharmacoki-
HER-2 positive breast cancer cell lines than PTX-NCs (Noh et al., 2016). netic and biodistribution profiles indicated that the coated NCs were
Shubar et al. investigated the role of apolipoprotein E (apo E) coat on rapidly removed from the blood stream. The authors hypothesized that
the surface of atovaquone nanocrystals (ANCs). It was observed that this was due to the shedding of PEGylated coating from the surface of
incubation of apo E coated ANCs with brain endothelial cells resulted in NCs (Polomska et al., 2017).
the accumulation of nanoparticles on the surface but not in their uptake In some cases, this shedding of stabilizers may be beneficial. For
into the brain cells (Shubar et al., 2009). Shegokar et al. developed instance, Liu et al. studied the shedding phenomenon by preparing PTX
nevirapine nanosuspension with surface modification by dextran, NCs stabilized with D-α-tocopheryl polyethylene gycol 1000 succinate
serum albumin, and polyethylene glycol to improve its targeting action. (TPGS). The rationale of this study was that TPGS acts as a P-gp in-
In vivo tissue distribution studies showed an increased NCs accumula- hibitor which on shedding improved the drug uptake in resistant cells.
tion in various organs like brain, liver and spleen thereby prolonging This was indeed demonstrated that in NCI/ADR-RES cells, over-
their residence at target site as compared to pure drug (Shegokar and expressing P-gp and PTX resistant, TPGS stabilized formulation pre-
Singh, 2011). Pandey et al. prepared docetaxel (DTX) nanocrystals and sented a distinctly improved anti-proliferative effect in comparison to
stabilized them with a novel chondroitin sulphate A (CSA) and poly- free PTX or poloxamer 407 stabilized PTX formulation (Liu et al.,
ethylene glycol congujate. This stabilizer provided multiple advantages 2010).
10
D. Patel, et al. International Journal of Pharmaceutics 586 (2020) 119555
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
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
tive. At the same time, drugs that are sparingly soluble could be good
Dose
nology aids in dealing with the technical hurdles of the aseptic pro-
Meloxicam
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. International Journal of Pharmaceutics 586 (2020) 119555
Declaration of Competing Interest Deng, J., Huang, L., Liu, F., 2010. Understanding the structure and stability of paclitaxel
nanocrystals. Int J Pharm. 390, 242–249.
Dogra, P., Adolphi, N.L., Wang, Z., Lin, Y.-S., Butler, K.S., Durfee, P.N., Croissant, J.G.,
The authors declare that they have no known competing financial Noureddine, A., Coker, E.N., Bearer, E.L., 2018. Establishing the effects of meso-
interests or personal relationships that could have appeared to influ- porous silica nanoparticle properties on in vivo disposition using imaging-based
pharmacokinetics. Nat. Commun. 9, 1–14.
ence the work reported in this paper. Dong, Y., Ng, W.K., Shen, S., Kim, S., Tan, R.B., 2009. Preparation and characterization of
spironolactone nanoparticles by antisolvent precipitation. Int. J. Pharm. 375, 84–88.
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