Importance of Standardizing Analytical Characterization Methodology for Improved Reliability of the Nanomedicine Literature

Abstract

Understanding the interaction between biological structures and nanoscale technologies, dubbed the nano-bio interface, is required for successful development of safe and efficient nanomedicine products. The lack of a universal reporting system and decentralized methodologies for nanomaterial characterization have resulted in a low degree of reliability and reproducibility in the nanomedicine literature. As such, there is a strong need to establish a characterization system to support the reproducibility of nanoscience data particularly for studies seeking clinical translation. Here, we discuss the existing key standards for addressing robust characterization of nanomaterials based on their intended use in medical devices or as pharmaceuticals. We also discuss the challenges surrounding implementation of such standard protocols and their implication for translation of nanotechnology into clinical practice. We, however, emphasize that practical implementation of standard protocols in experimental laboratories requires long-term planning through integration of stakeholders including institutions and funding agencies.

Full text

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Importance ofStandardizing Analytical
Characterization Methodology forImproved
Reliability oftheNanomedicine Literature
ShahriarSharifi1, NoufN.Mahmoud1,2,3, ElizabethVoke4, MarkitaP.Landry4,5,6,7*,
MortezaMahmoudi1*
HIGHLIGHTS
• The use of current standard protocols for robust characterization of nanotechnologies can significantly improve the reproducibility of
nanoscience data particularly for studies seeking clinical translation.
• The use of current standard protocols for robust characterization of nanotechnologies can significantly improve the reproducibility of
nanoscience data particularly for studies seeking clinical translation.
• Institutions, funding agencies, and publishing venues have a vital role in the practical implementation of the standard protocols of
nanomaterials characterization.
ABSTRACT Understanding the interaction between biological struc-
tures and nanoscale technologies, dubbed the nano-bio interface, is
required for successful development of safe and efficient nanomedicine
products. The lack of a universal reporting system and decentralized
methodologies for nanomaterial characterization have resulted in a low
degree of reliability and reproducibility in the nanomedicine literature.
As such, there is a strong need to establish a characterization system to
support the reproducibility of nanoscience data particularly for studies
seeking clinical translation. Here, we discuss the existing key standards
for addressing robust characterization of nanomaterials based on their
intended use in medical devices or as pharmaceuticals. We also discuss
the challenges surrounding implementation of such standard protocols
and their implication for translation of nanotechnology into clinical
practice. We, however, emphasize that practical implementation of standard protocols in experimental laboratories requires long-term
planning through integration of stakeholders including institutions and funding agencies.
KEYWORDS Characterization; Nanomedicine; Standard protocols; Reproducibility; Nanomedicine devices
e-ISSN 2150-5551
CN 31-2103/TB
PERSPECTIVE
Cite as
Nano-Micro Lett.
(2022) 14:172
Received: 23 June 2022
Accepted: 21 July 2022
© The Author(s) 2022
https://doi.org/10.1007/s40820-022-00922-5
Shahriar Sharifi, and Nouf N. Mahmoud have contributed equally to this work.
* Markita P. Landry, landry@berkeley.edu; Morteza Mahmoudi, mahmou22@msu.edu
1 Department ofRadiology andPrecision Health Program, Michigan State University, EastLansing, MI, USA
2 Faculty ofPharmacy, Al-Zaytoonah University ofJordan, Amman11733, Jordan
3 Department ofBiomedical Sciences, College ofHealth Sciences, QU Health, Qatar University, 2713Doha, Qatar
4 Department ofChemical andBiomolecular Engineering, University ofCalifornia, Berkeley,Berkeley, CA, USA
5 Innovative Genomics Institute, Berkeley, CA, USA
6 California Institute forQuantitative Biosciences, University ofCalifornia, Berkeley, CA, USA
7 Chan Zuckerberg Biohub, SanFrancisco, CA, USA

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1 Introduction
Nanomedicine is an umbrella term defined by the Ency-
clopedia Britannica as“a branch of medicine that seeks
to apply nanotechnology—that is, the manipulation and
manufacture of materials and devices that are roughly 1
to 100nm (nm; 1nm = 0.0000001cm) in size—to the
prevention of disease and to imaging, diagnosis, moni-
toring, treatment, repair, and regeneration of biological
systems” [1]. According to this definition, it is clear that
nanomedicine covers a broad range of medical products
ranging in purpose and performance requirements from
therapeutic nanoparticles containing drugs to be deliv-
ered in vivo with stringent control to nano-biosensors for
development of invitro/ex vivo diagnostics kits with lower
associated risk compared to injectable nanoformulations
[2–5]. In general, nanomedicine has shown significant
therapeutic success in both invitro and non-human in
vivo studies. However, successful clinical translation of
nanomedicines has lagged behind the successes implied
by numerous positive preclinical findings [6–8]. Unlike
the in vitro conditions in which most nanotechnologies
are validated, when nanoscale materials enter biological
environments, their physiochemical properties change
through the spontaneous adsorption and interaction of
the synthetic nanoscale material with the complex bio-
molecular environment, forming the nano-bio interface.
This nano-bio interface is incredibly complex, dynamic,
and difficult to characterize. While scientific reproduc-
ibility is an issue that plagues multiple fields of study [9],
the unique multidisciplinary nature of nanomedicine may
contribute to challenges of methodological reliability and
reproducibility issues. Nanomedicine combines expertise
from several fields including materials science, chemis-
try, biology, physics, pharmacology, and the like, and it
can therefore be particularly challenging to establish best
practices for collecting and reporting data when working
across multiple fields.
One can assume that if unreliability or lack of reproduc-
ibility [10–12] were the major barriers in nanomedicine,
few products would pass through the regulatory pipeline to
reach the market. However, an increasingly large number of
medical products using nanotechnology have reached the
market, comprising a multi-billion dollar industry, such as
the recently developed SARS-CoV2 vaccine formulations
by Moderna and Pfizer/BioNTech among others [13].
More generally, according to the FDA database of medical
devices, about 2586 different ’nano’ medical devices were
sold in the USA from 1980 to 2017 [14]. These nanomedi-
cal devices have mainly includedin vitrodiagnostic devices
including nanosilver or nanogold particles used ubiquitously
in ex vivo tests such as rapid at-home tests, orthopedic and
dental implants with nanostructure surfaces, wound dress-
ings including silver nanoparticles or nanofibers, bone void
fillers including nano-calcium phosphates, stents including
nanomaterial coating, vascular grafts including nanomaterial
coatings, or catheters coated with silver or other nanomate-
rials [15]. Conversely, a few of these nanomedical devices
consist of nanoparticulate formulations used for parenteral
and intravenous administration [16]. The list of commercial
FDA-approved nano-drugs, in which drugs are encapsulated
for delivery by nanoparticles, is quite short and limited to a
few well-established nanoscale systems such as lipid nano-
particles [17]. Herein, we describe the challenges to creating
“blanket” analytical and reporting guidelines for nanomate-
rials and nanomedicine research, the consequences thereof,
and some of the initiatives that have begun to take hold in
the field to establish standard guidelines.
2 Causes andSolutions toReproducibility
inNanomaterials Research andReporting
Several factors can contribute to poor methodological
quality and reproducibility of nanoscience publications,
independent of whether those nanotechnologies are sub-
sequently used in biomedical applications. Additionally,
the “file drawer” issue (i.e., the tendency to only publish
the positive results) in nanomedicine may misrepresent
actual findings and remains under-investigated [18]. More
broadly, a lack of nanoparticle characterization with a uni-
versally established quality control pipeline is considered
a primary challenge to building a cohesive body of lit-
erature in nanotechnology and makes it particularly chal-
lenging to predict how these nanotechnologies will fare
in biological environments. For instance, it is common to
assess nanomaterial properties such as the chemical com-
position, polydispersity index, size, charge, and other such
physiochemical properties invitro despite the intended use
of nanotechnologies invivo. Similarly, there is a lack of
standardization for what physiochemical properties should

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be assessed, and whether the reported variabilities rep-
resent average measurements over multiple experimental
replicates of the same sample (less rigorous) versus mul-
tiple technical replicates over independently synthesized
nanoparticle batches (more rigorous). Another common
issue is that solution-phase characterization methods such
as dynamic light scattering (DLS) are difficult to imple-
ment for non-spherical nanoparticles or highly polydis-
perse samples. For example, studies revealed significant
variations in the DLS results of the exact same type of
nanoparticles through different laboratories [19]. This lack
of universally established quality controls likely contrib-
ute to variable reproducibility outcomes in nanoscience.
These lacks of standardized guidelines are particularly
deleterious for nanoscience studies wishing to implement
nanotechnology in biomedicine. Another common theme
in nanomaterials research is a lack of thorough analytical
validation of nanoparticle surface chemistry. For instance,
carbon nanotubes (and numerous other types of nanopar-
ticles) have been the subject of a decades-long debate
on their biocompatibility, or lack thereof [20]. However,
many studies, particularly those claiming cytotoxicity,
lack analytical validation of nanoparticle purity and/or
surface chemistry [21]. Meanwhile, nanomaterial purity
can vary greatly from the specifications provided by the
manufacturer, such as commercially-procuredcarboxy-
lated nanotubes (COOH-SWNT) that were found to con-
tain over 80% more amorphous carbon contamination than
specified by its supplier, Sigma-Aldrich [22]. Therefore,
it is possible that reports of carbon nanotube toxicity may
originate from the toxicity of residual nanotube synthesis
by-products such as amorphous carbon and residual metal
precursor catalysts, or from intended or unintended modifi-
cations to nanotube surface chemistry [23].Similarly, tox-
icity reports of other nanomaterials may also be related to
impurities or bi-products in their production, rather than
the nanomaterial itself - a distinction that is not possible
to determine without detailed analytical characterization
of the studied nanomaterial samples.
Generally, it is common to see that the rigor and extent
of nanoparticle characterization is directly proportional to
the risk associated with the downstream use of that nano-
technology in biomedical applications. For example, the
Scientific Committee on Emerging and Newly Identified
Health Risks (SCENIHR) recommends a risk-based sys-
tem in which stringency of evaluation and characterization
of nanomaterials depends on nanomaterial type, duration
of contact with the body, and the nature of patient interac-
tion with devices (Fig.1) [24]. For instance, known bio-
incompatible materials that would interact directly with
human tissues and/or are likely to leak out of their devices
during use would be subject to more stringent characteri-
zation and biocompatibility testing than nanomaterials that
are known to be biocompatible and/or are unlikely to come
into direct contact with patients in clinical use. The char-
acterization of nanomaterials in scientific papers, however,
can vary widely from study to study since these materials
are mostly used in preclinical animal models. One com-
mon omission from many academic studies is analytical
assessments of nanomaterials when used as-procured from
commercial sources, despite large possible variabilities in
the quality and purity of commercially procured nanoma-
terials from supplier-to-supplier, from batch-to-batch, and
even from the vendor-provided spec sheet [22]. Efforts
to increase the rigor and reproducibility of nanomaterials
science research have been initiated both from the scien-
tific community and from national regulatory bodies as
described below.
3 Research Community‑driven Efforts
toIncrease Rigor andReproducibility
ofNanoscience Studies
To improve the reproducibility of nanoscience-based publi-
cations intended for use in biomedical applications, Caruso
and co-workersrecently proposed the use of a reporting
checklist, Minimum Information Reporting in Bio-Nano
Experimental Literature (MIRIBEL), as a requirement for
publication [21]. MIRIBEL checklist items include, for
example, details on material characterization (e.g., synthesis,
composition, size, shape, dimensions, size dispersity, and
aggregation), biological characterization (e.g., cell seeding
details, cell characterization, and passage), and experimental
protocol details (e.g., culture dimensions, administered dose,
method of administration, and delivered dose) [21]. Expert
responses from the nanomedicine community, despite sup-
porting the initial goals of the MIRIBEL checklist, dem-
onstrated the need for a list more closely tailored and con-
tinuously adjusted to criteria based on the intended use of
nanomedicines [10, 26]. In addition to the checklist, collect-
ing accurate and valid information on the characterization

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of nanomedicines using standardized methodologies is a
key step toward a more precise understanding and/or pre-
diction of the safety and therapeutic/diagnostic efficacies of
nanomedicine products. In other words, although reporting
minimum information related to type and results of char-
acterization is essential that is insufficient to guarantee
the repeatability and reliability of nanomedicine data. For
instance, many characterization techniques such as DLS or
zeta potential measurements are highly dependent on experi-
mental conditions and sample preparation details. Therefore,
reporting a value of physicochemical properties (e.g., size
via DLS) without mentioning data acquisition details (e.g.,
for DLS: type of medium employed, concentration of par-
ticles, type or size of cuvette, wavelength of laser, filtration
conditions, and data rendering: number count vs. raw inten-
sity) may result in discrepancies between laboratories on
Fig. 1 Risk evaluation of nanomedicine devices based on estimation of internal and external exposure. Based on the type of organ, duration of
exposure of nanomaterials, and their physicochemical characteristics, humans can be subject to negligible or high internal exposure to nanoma-
terials. The figure is drawn based on the data provided in the Scientific Committee on Emerging and Newly Identified Health Risks (SCENIHR)
report entitled “Guidance on the determination of potential health effects of nanomaterials used in medical devices” [25]

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identical nanoparticles. For example, it was shown that the
outcomes of two of the most commonly applied techniques
for nanoparticle sizing (i.e., DLS and differential centrifugal
sedimentation) of identical nanoparticles by different labo-
ratories were significantly different [19].
4 Nationally driven Efforts toIncrease Rigor
andReproducibility ofNanoscience Studies
On a national and international scale, regulatory agencies in
the USA and Europe have issued and established several sets
of rules, guidelines, and recommendations for evaluation of
nanomedicine products. For example, the FDA evaluates nano-
medicinal products on a case-by-case basis, employing the
combination product framework to determine the type of prod-
uct and resulting regulatory requirements for its review [5].
The FDA has identified several challenges and gaps related to
physicochemical characterization, biocompatibility, and toxic-
ity to evaluate nanoparticles that are incorporated into medical
devices and consequently has launched the Nanotechnology
Program in the FDA’s Center for Devices and Radiological
Health (CDRH). The program focuses on regulatory research
in evaluating the physicochemical properties and toxicity of
nanomaterials utilized in medical devices, and the impact of
the manufacturing processes on these properties. Similarly, EU
regulation such as 2017/745 specifically recommends highly
stringent conformity assessment procedures (Class III, highest
class risk in Europe) for medical devices containing nanoma-
terials, particularly when nanomaterials in those devices carry
a high likelihood of direct human exposure.
Considering these regulations and standards more closely,
standards such as ISO/IEC17025 “testing and calibration
laboratories” or good laboratory practice (GLP) as defined by
either the FDA or the European Council evaluate an organi-
zation’s technical competence in using analytical instruments
or testing to generate reliable and reproducible results [27].
Implementation of GLP requires establishing a quality assur-
ance team to verify and document compliance with GLP
rules, verification of the maintenance and calibration of ana-
lytical instruments, validation of analytical methods, as well
as development of standard operation procedures. Academic
settings or laboratories seldom implement all such standards
or regulations, likely due to a dearth of training on quality-
or GLP-related subjects [28]. It seems that GLP and similar
regulations such as GMP are ultimately established to protect
patient health and safety; the end-users of nanoscale medical
biotechnologies. However, the implementation of these regula-
tions in the academic research settings is limited. Academic
laboratories currently emphasize safe standard operating pro-
cedures implemented in the laboratory setting with a center on
experimenter and environmental health and safety. However,
implementation of GLP regulations that focus on rigor and
reproducibility of nanomedical devices in clinical setting in
academic research settings is not mandatory, since most aca-
demic research studies are not directly connected to patient
trials or clinical health outcomes. However, this lack of uni-
versal quality control in academic settings can make it difficult
to identify nanotechnologies that are the most promising pre-
clinical candidates and can lead to premature invivo testing of
sub-optimal nanomedicines or, conversely, lead to overlooking
promising nanotechnologies for invivo translation.
5 Non‑governmental Organization Standards
andGuidelines forNanomaterial
Characterization
Standards institutes and regulation agencies have provided
a number of documents and guidelines to address the
requirements of analytical characterization and validation
of nanomaterials. For example, one of the sets of available
standards for nanomaterial characterization was provided
by the International Organization for Standardization
(ISO; www. iso. org). ISO is a nongovernment organiza-
tion and network of national standards institutes in 162
countries, represented by one member in each country.
The ISO Technical Committee (TC) 229 was established
in 2005 standardize the process of creating and reporting
new nanoscale materials. The ISO (TC) 229 committee
has five working groups designed to develop standards for
terminology and nomenclature, measurement and charac-
terization, health and safety, materials specification as well
as product and application of nanomaterials [29, 30]. Until
now, the ISO (TC) 229 committee has published 100 stand-
ards related to nanotechnology, with 31 still under devel-
opment. The material-specific standards in the ISO (TC)
229 series are listed in Fig.2 which notably lists several
characterization methods for each class of nanomaterial.
In addition to material-specific standards (Fig.2), the ISO
and especially the ISO (TC) 229 have also developed other
standards for robust characterization of nanomaterials.

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Fig. 2 Examples of available material-specific standards and characterization methodologies for different nanomaterials. Documentation on
characterization methodologies for different nanomaterials is available based on ISO ID numbers and can help compile best practices for the
analysis and characterization of different nanomaterials

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For example, scanning electron microscope (SEM) and
transmission electron microscope (TEM) imaging are the
gold standards for characterizing the shape, size, and size
distribution of nanoparticles. The International Standard
ISO 19749 and ISO 21363 provide guidelines related to
sample preparation for powder and liquid samples, depo-
sition of nanoparticles on a substrate, number of samples
to be prepared and measured, number of particles to be
measured for particle size and shape determination, image
acquisition, data analysis, and reporting of results. The
standards report that uniform distribution of nanoparticles
with minimal aggregation across the entire measurement
substrate is essential for accurate analysis and reporting
of particle shape, size, and size distribution. Measure-
ment errors can arise with aggregated particles, such as
aggregated particles being counted as one particle. The
current standards present essential sample preparation
techniques such as powder and liquid sample deposition
techniques with minimum agglomeration, selecting a
suitable measurement substrate that enhances the contrast
between particle and background, and the adhesion of the
particles across the substrate, optimizing the concentration
of the sample, drying methods, and use of representative
samples. It is important to note that these optimizations
are often user-defined and thus subject to human biases.
Furthermore, the quality of TEM and SEM images are
strongly dependent on several factors including sample
preparation, nanoparticle size, nanoparticle stability under
electron beam exposure, and nanomaterial atomic mass.
6 Efforts Implemented toIncrease
Nanomaterials Science Reproducibility
The establishment of above-detailed standards represent an
important step forward for the nanoscience community yet
implementing these standards and enforcing their use for
publication remains sporadic. Currently, thorough charac-
terization of nanomaterials using several orthogonal ana-
lytical techniques is considered best practice in the nanosci-
ence community. Numerous techniques are often available
to analyze each physiochemical property, and selection of
which analytical techniques used to characterize nanoparti-
cle samples are often based on their eventual intended use
in nanomedicine [11]. Furthermore, an important compo-
nent of GLP requirements includes ensuring that analytical
methods are properly calibrated to ensure that their perfor-
mance meets the requirements for the intended applications.
According to USP (U.S. Pharmacopeia),the analytical char-
acteristics of a method including accuracy, precision, speci-
ficity, detection limit, quantitation limit, linearity, range, and
robustness need to be identified and validated prior to sample
characterization, such that different batches and altogether
different samples can be compared to each other and against
the same known baseline. Conversely, many published stud-
ies focus on the formulation of drug nanocarriers and dem-
onstrate the formulation’s intended efficacy in an animal
model [31, 32]. Often overlooked are the quantification of
residual or precursor materials in commercially procured
starting nanomaterials and aforementioned standardization
of analytical calibrations [33–37]. For instance, the valida-
tion requirements for analytical methods such as high-per-
formance liquid chromatography for pharmaceutical analysis
and quantification of active pharmaceutical ingredients in
the presence of nanocarriers are already established [38] and
implemented for clinical studies. Nevertheless, validation
for most analytical techniques used in the characterization
of nanomaterials or carriers such as DLS, zeta potential,
SEM, or TEM is not a common practice [39]. Hence, there
is great potential risk for nonstandard or uncontrolled ana-
lytical processes to compromise the methodological qual-
ity and, consequently, the reliability of published research
works especially in the field of nanomedicine.
7 Existing Standards andGuidelines
forNanomaterial Biocompatibility
Assessments
The ISO and especially ISO (TC) 229 have also developed
several standards for biological and toxicity evaluation of
nanomaterials (Fig.3a). For example, the ISO/TR 16196 and
ISO/TR 16197 standards discuss essential protocols on sam-
ple preparation and dosimetry of nanomaterials for reliable
toxicological screening. The Technical Specification ISO/
TS 19337:2016(E) describes the characterization of working
nanomaterial suspensions when conducting invitro toxicity
assays and applicable measurement methods. The techni-
cal report presents a useful staged scheme for performing
measurements (Fig.3b). For working stock suspensions of
nanoparticles, the presence of endotoxins should be veri-
fied, since they may significantly alter the invitro toxicity

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Fig. 3 Examples of standards related to the invitro biological tests and toxicity evaluations/monitoring of nanomaterials. a ISO (TC) 229 com-
mittee standards provide general guidelines and recommendations for nanoparticle characterization in biological environments, and for evalu-
ation of nanoparticle biocompatibility in living systems. Standard labeled by “*” are currently under development. b Schematic showing the
states at which measurements are made (Technical Specification ISO/TS 19337:2016(E))

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test results. Furthermore, other characterizations of nano-
particles that may affect the toxicity assay results should be
performed and reported. For example, the working suspen-
sions’ stability, by measuring particle size and concentra-
tion as a function of time, should be assessed both invitro
and, in an environment, mimicking its intended use (i.e., in
plasma if to be injected into the bloodstream). This level of
analysis in biofluids is necessary because upon introduction
of nanoparticles into biofluids, the surface of nanoparticles
is rapidly coated with constituents of biofluids such as lipids
and proteins, forming the nanoparticle protein corona [40].
Another important characterization to undertake and report
is the concentration of metal ions in nanoparticle samples,
where many nanomaterials are synthesized with the use of
metal precursors, whose residual presence may not always
be accurately reported by manufacturers but can significantly
affect toxicity outcomes.
Another ISO committee, ISO/TC 194 Biological and
Clinical Evaluation of Medical Devices, has also developed
standards and guidelines for characterization and develop-
ment of nanomaterials used in medical devices. The ISO/
TC 194 committee produced the important ISO 10993–22,
which compiles and disseminates the safety and biocompat-
ibility of various nanomaterials [41]. This standard describes
the characterization of nanomaterials and, in line with ISO/
TR 13014, “guidelines for physicochemical characterization
of nanomaterials,” provides a framework including a series
of considerations and recommendations to improve the qual-
ity and reproducibility of nanomaterials’ characterization
and evaluation. The ISO/TR 13014 standard recommends
that some basic properties such as chemical composition,
purity, object size and size distribution, aggregation and
agglomeration state, shape, surface area, surface chemis-
try, surface charge, solubility, and dispersibility need to be
assessed, and, based on the type of nanomaterial and its
intended use, additional characterization such as redox
potential, radical formation potential, or crystallinity may
also be needed. Moreover, the ISO/TR 13014 standard also
mentions specific nanomaterial characterization methods
to be routinely undertaken including DLS, SEM, and zeta
potential measurements. In addition, the technical report
ISO/TR 10993–22 describes several aspects of cytotoxicity
evaluation and compatibility of nanomaterials specifically
intended to be used in conjunction with existing medical
or clinical diagnostic devices. Because nanomaterials can
have broad absorption properties across the electromagnetic
spectrum, they can interfere with standard medical or diag-
nostic assays which use standard dyes or fluorophores.
These additive optical interactions can lead to variable
results, artifacts related to nanomaterial-dye interactions,
and overlooked cytotoxicity. Therefore, appropriate controls
and removal of nanoparticles by centrifugation or filtration
before reading results can minimize artifacts and reduce
variations in the results. Furthermore, the technical report
ISO/TR 10993–22 discusses guidelines for general genotox-
icity, carcinogenicity, reproductive toxicity, immunotoxicity,
irritation and sensitization, and hemocompatibility related to
the use of nanoparticles in clinical testing settings.
As stated above, the extent of nanomaterial characteriza-
tion performed is often based on the risk associated with
the product. Intended use is critical for defining the required
information to be reported in the experimental literature to
ensure reproducibility. For example, nanopharmaceuticals
have inherently higher risk of negative biocompatibility
outcomes compared to non-implantable medical devices, as
their mode of action is through direct contact with a patient
and can interfere with immunological, pharmaceutical, or
metabolic pathways. The formulation, type of application,
pharmacokinetics, as well as dosage of nanomedicine prod-
ucts can also define the approach to their characterization.
For example, unlike nanomaterials used in pharmaceuticals,
which are usually in colloidal form, nanomaterials used in
medical devices can have various forms. Due to this variety
in dosage and forms, the ISO/TR10993-22 also provides
classifications for nanomaterials used in medical devices,
either as surface-bound nanostructures, which are to be
incorporated within a medical device with or without the
intention of being released, versus nano-objects that might
be released from a medical device as a product of degrada-
tion, versus medical devices that are themselves nanoscale
objects (Fig.4). Proper knowledge and identification of
nanomaterials’ physicochemical characteristics and bio-
compatibility prior to incorporation into medical devices
are essential to understand their compatibility with other
composites and determine the final product’s biocompatibil-
ity and toxicological effects [42]. In addition, nanomaterials
used in medical devices or nanopharmaceuticals will ulti-
mately be exposed to biological media containing body flu-
ids; hence, the above-mentioned parameters must include the
effect of the protein corona [41]. Depending on the type of
nanomaterials and whether they will be static on the device

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or released, the ISO/TR 10993-22 has several recommenda-
tions for safety evaluation of devices.
Based on the intended use of nano-based devices (e.g.,
for orthopedic implants versus exvivo diagnostics), char-
acterization of additional nanomaterial features should be
undertaken. For example, the surface topography of implants
has been shown to have critical influence in modulating the
immune response as it can provoke inflammation and foreign
body reactions [43]. In 2019, the FDA recalled highly tex-
tured breast implants due to risk of breast implant-associated
anaplastic large cell lymphoma, a cancer of the immune sys-
tem [44]. In this example, interaction of cells with these
nanostructures, possibility through disintegration or break-
age of these breast implant topological features, motivated
standardization of nanomaterial-based implants regarding
assessment of their degradation or dissolution and measure-
ment of average surface roughness.
Although the above-mentioned standards will not cover all
types of nanomaterial-based or nanomaterial-incorporated
medical devices, nor all toxicity assessments, they address
many critical requirements and especially characterization
criteria and reporting requirements for nanomaterials used in
the clinic. In addition to the surface topology example above,
these standards cover various aspects of invitro biological
testing, sample preparation, and interaction of the nanoma-
terials with biofluids relevant to their intended use (Fig.4).
It is worth to note that demonstration of compliance to these
standards is a regulatory requirement for product approval
and market launch.
Fig. 4 Characterization requirements for medical devices containing nanostructures and nanomaterials as recommended by ISO/TR 10993–22.
The extent of characterization is dependent on the type and state of nano-based medical devices. The nanomaterial exposure risk via direct con-
tact or unintended nanoparticle leakage from the device needs to be considered in the device characterization to properly assess safety and effi-
cacy of nanotechnology-based medical devices. The degradation or dissolution and stability of nanostructures in relevant biological media need
to be monitored and characterized over the shelf life and active lifetime of medical devices. Finally, the structures need to be fully characterized
both invitro and in invivo proxies to ensure the design and physicochemical properties do not compromise the safety and efficacy of the medical
devices. The scrutiny of the evaluation will increase if the nanostructures are designed to release from the device or pose the risk of undesired
release in biological fluids. In addition to the above-mentioned evaluations, further tests (e.g., biodistribution, toxicity, and release kinetics of
nanomaterials) are required to ensure the nanomedical device is safe for use in the clinic. The ISO/TR 10993–22 standards provide a framework
and guidelines for characterization of nanomaterials. More specific physicochemical characteristic testing of nanomaterials is detailed in ISO/TR
13014. The figure is drawn based on the information provided in ISO/TR 10993–22 [41]

Nano-Micro Lett. (2022) 14:172 Page 11 of 15 172
1 3
It is also noteworthy that standardization efforts for nano-
material characterization are not limited to the ISO com-
mittee. The American Society for Testing and Materials
(ASTM), another internationalstandard organization, has
a committee on nanotechnology (E56) which coordinates
the nanotechnological need and addresses issues related
to standards and guidelines related to nanotechnology
and nanomaterials. ASTM E 56 has several subcommit-
tees which include standards oninformatics and terminol-
ogy (E56.01), physical and chemical characterization of
nanomaterials (E56.02),environment, health, and safety of
nanomaterials (E56.03), nano-enabled consumer products
(E56.06), education and workforce development (E56.07)
and nano-enabled medical products (E56.08). The impor-
tance of training, especially regarding selection and opera-
tion of nanotechnology infrastructure, is critical as it directly
affects the precision and accuracy of nanotechnology data
and results. Hence, while ASTM standards cover a variety of
nanotechnology characterization techniques, those standards
place a particularly strong emphasis on education and train-
ing in the field. For example, ASTME3001-20 describes a
procedure for education and training on the use and analysis
of characterization methods for nanometer-scale materials.
The E56 and specially E56.08 also have a few guidelines
and standards for medical products that have nanoscale
features or use nanoparticles. For example, three standards
address lipid quantification in liposomal products (E3297-
21, E3323-21, and E3324-22). Similar to ISO, ASTM has
also developed standards for the characterization of nano-
particles as mentioned in E56.02.
Other agencies such as the EuropeanNanomedicineChar-
acterization Laboratory (EUNCL) and the US National Can-
cer Institute Nanotechnology Characterization Laboratory
(NCI-NCL) have jointly developed several standard operat-
ing procedures (SOPs) for characterization and assessment
of nanomaterials used in medicine. These SOPs include
size and size distribution, concentration, surface chemistry,
chemical composition, invitro assays (e.g., immunologi-
cal, toxicity, oxidative stress), and invivo assays protocols
[45, 46]. The EUNCL and NCI-NCL have jointly developed
multiple standard operating procedures (SOPs) for nanopar-
ticle analyses which address method validation ranging from
proper sample preparation to calibration requirements. For
example, the Joint Assay Protocol, PCC-1, published by
EUNCL and NCI-NCL details experimentation conditions
that should be used for DLS measurements. This protocol
specifies that the typical nanoparticle sample concentration
for DLS measurements is 1mg/ml, but needs to be modi-
fied according to the scattering properties of the sample.
The hydrodynamic size measured by DLS is also noted to
depend on the salt concentration of the suspending medium,
and the guidelines recommend performing DLS measure-
ments using supporting inert monovalent electrolytes (e.g.,
10mmol NaCl) and to avoid using pure deionized or dis-
tilled water which can cause a decrease in the measured
diffusion coefficient and an apparent increase in hydrody-
namic size. Furthermore, the nanomaterial sample and the
dispersion medium should be filtered before measurement to
prevent background scattering due to dust or contaminants.
Lastly, the refractive index of the dispersion media should
be measured and included for the subsequent calculations of
the sample diffusion coefficient.
The above protocol also highlights the minimum report-
ing requirements mentioned in ISO 13321:1996 (now ISO
22412:2017) that include: particle concentration (mass or
volume based), dispersion medium composition, refractive
index values for the particles and the dispersion medium,
viscosity value for the medium, measurement temperature,
filtration or other procedure used to remove extraneous
particulates/dust prior to analysis (including pore size and
filter type), cuvette type and size (path length), instrument
make and model, scattering angle(s), and laser wavelength.
Few research papers report all these values in the nano-
technology literature [47].
Over recent years, there have been significant efforts
to uncover the reasons behind lack of reproducibility in
the nanomedicine literature and to identify strategies to
improve the robustness and accuracy of both characteri-
zation data and methodological approaches in nanomedi-
cine. Aside from using checklists (e.g., MIRIBEL), the
critical role of aforementioned standards is in addressing
robust characterization of nanomedicines based on their
intended use in medical devices and pharmaceuticals.
The main issue with regard to the use of available stand-
ards in academic laboratories, however, is the challenge
of “requiring” their implementations in the nanoscience
literature. Peer reviewers, editors, and journal owners/pub-
lishers have historically seldom requested the details of
standards or methods development in fundamental nano-
materials science, in the absence of biological applications
[29], unlike standardization checklists often required for
biological studies that mandate reporting of biological

Nano-Micro Lett. (2022) 14:172 172 Page 12 of 15
https://doi.org/10.1007/s40820-022-00922-5
© The authors
replicates, effect size calculations, ANOVA or other sta-
tistical significance calculations, antibody validation, plot-
ting of all data points for N < 10 instead of averages, dis-
closure of error type (SD, SE, CI), and other standardized
metrics by certain journals [48]. One critical challenge
to similar standardization requirements for the nanoma-
terial and nano-bio interface literature is in the time lag
between the inception of nanomaterials and nanotechnol-
ogy as a relatively young field of study, and the time it
takes to establish experimental and reporting standards.
Another challenge is a lack of in-depth training expected
or needed for undertaking studies in nanomedicine owing
to the interdisciplinary nature of nanoscience. With mul-
tiple fields coalescing in nanobiotechnology, the experi-
mental intuition and reproducibility standards that build
from many years training in a specific field can be lacking.
For example, using SEM and TEM for nanoscale imag-
ing began as early as the 1930s according to the national
nanotechnology initiative (NNI); however, the ISO stand-
ards related to characterizing nanomaterials by SEM (ISO
19749) and TEM (ISO 21363) were not published until
2021 and 2020, respectively. Similarly, recommendations
by EUNCL and NCI-NCL are also relatively new, provided
in the past ten years.
8 Call toAction
It is well-documented that the nanomedicine literature suf-
fers from poor reproducibility. In this paper, we have dis-
cussed one of the major causes of low reproducibility, stem-
ming from overlooking available standard characterization
methodologies, as the academic nanomedicine community
may not fully aware of these standards.
Methods to characterize nanomaterials are complex, and
various parameters can influence the accuracy and precision
of the results. Although there is an urgent need to resolve
the repeatability issue, there is no quick solution to this issue
especially when assessing the magnitudeand root causes
of irreproducibility remain incomplete. Implementation of
the discussed standards may seem to be a practical solution;
however, the implementation process can be challenging
considering the complexity and variability of methods asso-
ciated with nanotechnology and therefore requires involve-
ment of various stakeholders including research institutes,
journal editors, and funding agencies. While we do not
reiterate the technical details of the standardized protocols
described herein, we emphasize the importance of incorpo-
rating these standards into research practice and commu-
nicating their importance with trainees in the nanoscience
research community.
One technique highlighted here was DLS as it is the
most commonly used instrument for nanoparticle size char-
acterization in the literature. For so-called simple DLS
measurements, many parameters can affect the reliability
and repeatability of the measurement. Parameters such as
particle concentration (mass or volume-based), dispersion
medium composition such as salt concentration, refractive
index values for the particles and the dispersion medium,
viscosity value for the medium, measurement temperature,
filtration, or other procedure used to remove extraneous par-
ticulates/dust prior to analysis (including pore size and filter
type), cuvette type and size (pathlength), instrument make
and model, scattering angle(s), and laser wavelength all can
significantly affect the quality of the data from DLS. How-
ever, we could not identify manuscripts that had used DLS
characterization that also reported these values and param-
eters, highlighting the issues at play that affect the ability to
interpret DLS data and compromise the ability to reproduce
the results in peer-reviewed literature.
9 Conclusions
Herein, we have discussed the status of experimental vali-
dation standards and reproducibility in the nanoscience
community, with implications for the translation of these
nascent nanotechnologies into clinical practice. We hypoth-
esize that one main contributing factor to the lack of rigor
and reproducibility in nanoscience is due to different types
of nanotechnologies, each of which is unique in its physi-
ochemical and biocompatibility profiles and intended for a
wide variety of downstream biomedical applications. We
highlight that proper implementation and reporting of stand-
ards for characterization and methodology in nanomedicine
need to be the subject of careful collaborative investigation
and to be developed and broadly adopted by involved stake-
holders, e.g., researchers, funding agencies, and journal edi-
tors, and provide examples of efforts toward these goals.
Increasing awareness of the presence of established proto-
cols and standards—and more importantly, implementing

Nano-Micro Lett. (2022) 14:172 Page 13 of 15 172
1 3
these standards in nanomedicine research—may improve the
scientific rigor and reproducibility of nanoscience as applied
to biomedicine and allow quantitative comparisons of results
obtained by its various stakeholders.
Acknowledgements We acknowledge support from the U.S.
National Institute of Diabetes and Digestive and Kidney Diseases
(Grant DK131417) (MM). We also acknowledge support of a Bur-
roughs Wellcome Fund Career Award at the Scientific Interface
(CASI) (MPL), a Dreyfus foundation award (MPL), the Philom-
athia foundation (MPL), an NIH MIRA award R35GM128922
(MPL), an NIH R21 NIDA award 1R03DA052810 (MPL), an
NSF CAREER award 2046159 (MPL), an NSF CBET award
1733575 (to MPL), a CZI imaging award (MPL), a Sloan Founda-
tion Award (MPL), a USDA BBT EAGER award (MPL), a Moore
Foundation Award (MPL), and a DOE office of Science grant
DE-SC0020366 (MPL). MPL is a ChanZuckerbergBiohub inves-
tigator, a Hellen Wills Neuroscience Institute Investigator, and an
IGI Investigator.We alsoacknowledge support from aFulbright
fellowship(NNM).
Funding Open access funding provided by Shanghai Jiao Tong
University.
Declarations
Conflict of interest Morteza Mahmoudi discloses that (1) he is a co-
founder and director of the Academic Parity Movement (www. parit
ymove ment. org), a non-profit organization dedicated to addressing
academic discrimination, violence and incivility; (2) he is a Founding
Partner at Partners in Global Wound Care (PGWC); and (3) he receives
royalties/honoraria for his published books, plenary lectures, and li-
censed patent. The author declares no conflicts of interest.
Open Access This article is licensed under a Creative Commons
Attribution 4.0 International License, which permits use, sharing,
adaptation, distribution and reproduction in any medium or format,
as long as you give appropriate credit to the original author(s) and
the source, provide a link to the Creative Commons licence, and
indicate if changes were made. The images or other third party
material in this article are included in the article’s Creative Com-
mons licence, unless indicated otherwise in a credit line to the
material. If material is not included in the article’s Creative Com-
mons licence and your intended use is not permitted by statutory
regulation or exceeds the permitted use, you will need to obtain
permission directly from the copyright holder. To view a copy of
this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/.
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