Keywords
micromedicines - nanomedicines - quality control - guidelines - regulatory authorities
Introduction
Compared with traditional medicines, micro/nanomedicines have significant advantages
in terms of improved bioavailability, sustained release, targeted transportation,
and reduced toxicity. Micro/nanomedicines have received extensive attention from developers
and drug manufacturers due to their great development potential and broad application
prospects. An increasing number of micro/nanomedicines are entering the clinical trial
stage. However, few products have been successfully marketed. One of the reasons may
be a lack of comprehensive control over the quality of micro/nanomedicines.[1] Reliable characterization methods that reflect quality-related variations in in vivo performance lay the foundation for optimizing micro/nanomedicines and are beneficial
for reducing trial failure and the waste of human, material, and financial resources.
Compared with traditional medicines, the quality control of micro/nanomedicine is
more complicated. In the manufacturing process, even a slightest deviation can cause
a great change in their in vivo performance. Drug regulatory agencies in the United States, China, and the European
Union have issued guidelines on quality control of micro- and nanomedicines. All three
agencies recognize the particularities of nanodrugs and emphasize strict quality control
and assessment of their physicochemical properties, manufacturing processes, stability,
and safety to ensure the efficacy and safety of nanomedicines. Differences in the
quality control requirements among pharmaceutical regulatory agencies are summarized
in [Table 1].
Table 1
Differences in the quality control requirements for micro- and nanomedicines among
pharmaceutical regulatory agencies in China, the United States, and Europe
|
Agency
|
Differences
|
|
FDA
|
1. Has a more in-depth understanding of nanoproducts, considering products with a
particle size of 1–100 nm or those whose physicochemical properties or biological
effects change due to particle size variation as nanoproducts
2. Currently has industrial guidelines for nanotechnology in food, cosmetics, and
liposomes, but they are not mandatory
|
|
EMA
|
1. Places greater emphasis on risk prevention rather than purely technical assessments
2. The risk assessment of nanoproducts includes four parts: hazard identification,
hazard characterization, exposure assessment, and risk characterization
3. Currently has regulations for nanotechnology in cosmetics, nanomaterials, and food,
and is still developing comprehensive regulations and guidelines for the complexity
of nanodrugs
|
|
CDE
|
1. Provides more specific and detailed technical guidance principles, focusing on
the quality control research of nanodrugs
2. Emphasizes the full-process quality control of nanodrugs, including raw materials,
excipients, and packaging materials, as well as production, transportation, clinical
preparation, and usage stages
3. For the stability study of nanodrugs, more detailed requirements are proposed,
including stability during storage, preparation, and clinical use, as well as influence
factor investigations
|
Abbreviations: CDE, Center for Drug Evaluation; EMA, European Medicines Agency; FDA,
Food and Drug Administration.
According to the “Technical Guidance for Quality Control Study of Nano Drugs (interim))”
issued by the Chinese Center for Drug Evaluation (CDE), the classification of nanomedicines
is as follows: nanosized drug particles, carrier-based micro/nanomedicines, and other
nanomedicines. Among them, carrier-based micro/nanomedicines are the most prevalent,
and quality control is also the most complicated. The present review will focus on
the quality control of these carrier-based micro/nanomedicines. Carrier-based micro/nanomedicine
refers to the use of natural or synthetic polymers, lipid materials, proteinaceous
molecules, and inorganic materials (metabolically excreted) as carrier materials for
drug delivery, and based on a specific preparation process, active pharmaceutical
ingredients (APIs) are encapsulated, dispersed, noncovalently or covalently bound
to micro/nanocarriers to form particles of micro/nanoscale, such as microspheres,
liposomes, and micelles. The quality control indicators of carrier-based micro/nanomedicines
can be divided into two categories: the basic characteristics of formulations and
the nano-specific characteristics.[2] The basic characteristics of formulations include the identification of active ingredients,
content determination, related substances, and quality evaluation indicators required
by pharmacopeias for different dosage forms. For example, the pH, viscosity, osmotic
pressure, bacterial endotoxins, sterile, and insoluble particles are required for
injection, whereas weight differences, disintegration time limits, in vitro dissolution or release profiles, and microbial limits are required for oral solid
preparations.[3] The nano-specific characteristics include in vitro dissolution and release of the drug, particle size and distribution, structure and
morphology, surface properties, form and state of drug presence, encapsulation rate
and drug loading, stability, and critical micellization concentration.
In this review, we summarize the characterization methods commonly used for most nanocarrier
drug delivery, in accordance with national pharmacopeias, relevant guidelines, and
the literature. In addition, We also discuss special considerations and requirements
during quality control of different types of drug delivery systems (e.g., microspheres,
liposomes, micelles).
Nano-Specific Characteristics of Carrier-based Micro/Nanomedicines
Nano-Specific Characteristics of Carrier-based Micro/Nanomedicines
In vitro Release
In vitro release is an indicator to ensure consistency (batch-to-batch or generic-to-originator)
or to compare the advantages and disadvantages of each formulation during drug manufacturing.
More importantly, it may reflect the in vivo dissolution or release behavior of carrier-based micro/nanomedicines, reducing the
failure rate of clinical trials. Therefore, it is of great significance to establish
the in vitro release methods with strong predictability. Separation of loaded and released drugs
is essential to measure the release pattern of carrier-based micro/nanomedicines.
Currently, the most discussed evaluation methods include dialysis, sampling and separation,
flow cell, adaptive perfusion, and in situ methods. The small size of micro- and nanomedicines makes it difficult to separate
them from dissolved drug molecules. Even if separation is achieved, the presence of
dissolved drug molecules in low concentration renders them undetectable by conventional
analytical instruments. Regarding the standard in vitro dissolution methods, multiple rounds of sampling and replenishment of the blank medium
may result in the early removal of unreleased micro/nanomedicine, leading to poor
results. The burst release of micro/nanomedicine places greater demands on assessing
its accuracy and discrimination. However, there is no definite approach for evaluating
the degree of in vitro release of microplastics/nanomedicine. The methods developed by researchers in-house
exhibit deficiencies in terms of scientific rigor and standardization, which limits
the development and application of carrier-based micro/nanomedicines.
Sample and Separation Methods
Sample and separation methods refer to the dispersion of carrier-based micro/nanomedicines
directly in a release medium at constant temperature and sampling at predetermined
time points. Afterward, the drug is separated by ultrafiltration, ultracentrifugation,
or centrifugal ultrafiltration for measurement.[4] The multiple sampling steps can lead to drug loss. Centrifugation/ultrafiltration
may impair the carriers and lead to leakage of the loaded drugs. Other factors influencing
the method accuracy include media temperature, agitation speed, and media type.[5]
[6]
[7]
[8] The in vitro release of extended-release injection of aripiprazole (microspheres) and extended-release
injection of paliperidone palmitate (microcrystals) employs sample and separation
methods, which has been included in the Dissolution Database of Food and Drug Administration
(FDA).[9]
Dialysis-Based Methods
Dialysis-based methods have been extensively used for the in vitro release study of micro/nanomedicine. The method lies in the fact that free drugs
in the form of small molecules can permeate through the dialysis membrane, while micro/nanomedicine
cannot because of the relatively large particle size.[10] Therefore, the separation of micro/nanomedicine and free drugs can be achieved.
The drug release rate is determined by analyzing the concentration of free drugs.
Dialysis methods mainly include normal dialysis and reverse dialysis.[11]
In the normal dialysis method, the suspension of the micro/nanomedicine is sealed
in a dialysis bag, which is immersed in the release medium. Under magnetic stirring,
samples are taken from the medium at defined time points for measurement. The dialysis
method can be integrated with dissolution devices such as being tied to the paddles
or put in the baskets.[12] Due to the impeded diffusion of the drug in the dialysis bag and the lack of agitation
of the internal medium, the method may be unable to meet the sink condition, leading
to poor prediction of the in vivo release of intravenously injected nanomedicines such as liposomes, micelles, and
nanoemulsions.[13]
[14] However, the method may be applied to microspheres by mimicking the subcutaneous
or intramuscular environments.[15]
In the reverse dialysis method, the micro/nanomedicine is dispersed in the medium
outside the dialysis bag. The dialysis bag inside contains the release medium to receive
the released drug molecules. Since sink conditions can be met, this method is applicable
to carrier-based micro/nanomedicines that are administered intravenously or orally.[16] However, the dilution of the released drug can lead to stability concerns and challenges
in accurately measuring the burst release.
The advantage of the dialysis method is the convenience of sampling and changing the
release medium. The disadvantage lies in the adsorption and clogging of drug molecules,
biasing the measurement results. Therefore, the National Nanotechnology Centre does
not recommend the dialysis method for protein-containing micelles.[17] The key factors influencing the accuracy of the dialysis method include the molecular
weight cutoff of the dialysis membrane, the ratio of the medium inside and outside
of the dialysis bag, the composition of the medium, the agitation speed, and the temperature.[18]
There are already many commercialized dialysis devices available for in vitro release studies of nanomedicines, e.g., dialysis kits like Float-A-Lyzer G2 and Slide-A-Lyzer,
as well as commercialized dialysis chambers like Side-Bi-Side diffusion chambers.
The cellulose ester membrane used in the Float-A-Lyzer G2 is a synthetic membrane
with low protein adsorption. The tubular membrane tube prevents sample dilution. The
membrane tube is tipped with a screw-on cap that can be opened to facilitate sample
loading and sample recovery using a pipette tip and to avoid the risk of needle puncture.
Slide-A-Lyzer Dialysis Cassettes are constructed from two sheets of low-binding, regenerated-cellulose
dialysis membrane that is hermetically sealed on either side of a silicone-like gasket
inside an inert plastic frame. A flat cassette chamber with two membranes provides
a high surface area-to-volume ratio that maximizes diffusion rate compared to cylindrical
dialysis tubing. Rectangular cassette design maximizes recovery of the entire sample
volume via any one of the four corner injection ports.
Side-Bi-Side is a horizontal diffusion cell device. The dialysis membrane can be mounted
in the middle of the two diffusion cells and secured with fixing clips. A certain
amount of micro- and nanodrugs are placed in the supply chamber and the release medium
is placed in the receiving chamber, and the drugs can be transferred to the release
medium in the receiving chamber through the dialysis membrane layer, and the stirring
speed and medium temperature need to be controlled during the release process. The
advantage is that the device cost is low, the operation is convenient, and there is
no need for filtration and separation after sampling so that drug loss can be avoided.
The disadvantages are that the stirring effect is difficult to ensure, and the air
bubbles are difficult to remove, which may lead to uneven distribution of the released
drug in the receiving chamber.
The Flow Cell Method
The method utilizes Flow-Through Cell Apparatus (USP Apparatus 4) to measure the dissolution,
where a constant flow pump pulls the release medium to contact the drug. A filter
is positioned at the inner top of the cell to retain undissolved material, while the
concentration of the released drug can be measured.[19] Since the drug formulation is always exposed to a fresh medium, sink conditions
are well maintained. The method is used to determine the dissolution/release of micro/nanomedicine
administered intravenously or orally. One drawback is the low drug concentration in
the release medium, requiring a sensitive analytical method. Filters, on the other
hand, are susceptible to adsorption or clogging. The main factors affecting the flow
cell method include the flow rate and pattern of the release medium.[20] When the flow rate of the release medium is low, it may lead to a slow or even incomplete
release of a drug. The flow pattern includes laminar and turbulent flow. Turbulent
flow is induced in the absence of glass beads at the bottom of the cell, simulating
the gastrointestinal peristalsis in the fasting state; conversely, laminar flow is
formed to simulate a satiety state and prevent drug aggregation.[21]
Adaptive Perfusion
Adaptive perfusion is a new method recommended by the FDA. The adaptive perfusion
method is a pressure-controlled separation technique based on tangential flow filtration.
It enables size-based separation of particulates in drug products by adjusting the
filter cutoff, feed flow rate, or back pressure. This approach allows simultaneous
analysis of the drug released and residual drug in particulate drug systems, such
as emulsions, suspensions, liposomes, and drug-protein complexes. The method provides
faster drug release, e.g., minutes instead of hours, and higher release percentages
(>60%) compared with dialysis. Besides, adaptive perfusion is not limited by diffusion
through membranes, providing more sensitive drug release profiles.
Online Detection
Electrochemical detectors, turbidimetric, or laser diffraction can be combined with
dissolution instruments to achieve rapid, real-time, and online detection of drug
release in vitro.[22] Sample collection and separation are omitted. Electrochemical detectors monitor
the concentration of released electroactive drugs via ionic or drug-selective electrodes,
and as a result, this method does not apply to the analysis of nonionizing drugs.
Turbidimetry and laser diffraction methods determine the concentration by measuring
the light intensity passing through the drug release medium. However, these two methods
require a long equilibration time, a high sample concentration, and a suitable particle
size range.[4]
In vitro Release Conditions
To achieve in vitro-in vivo correlation, the simulation of the physiological environment by in vitro release conditions should be focused. Currently, the commonly used release media
to simulate the blood environment, in addition to adding 10% fetal bovine serum, 0.1%
Tween 80, and human serum, also included phosphate buffer (pH 7.4) or HEPES buffer
(pH 7.4).[23] For passive-targeted liposomes, researchers have established a two-stage reverse
dialysis method for in vitro release assays.[24] In the first phase, liposomes were dialyzed in HEPES buffer at pH 7.4 to simulate
the in vivo circulation of liposomes, whereas in the second phase, the liposomes were dialyzed
in HEPES buffer containing 1% Triton X-100 to simulate the drug release process at
the target site. The prepared liposomes consisted of lipids with a high phase transition
temperature and cholesterol, and no drug leakage was observed in the first phase of
the experiment. In the second phase, the drug release rates of liposomes with different
compositions differed significantly, demonstrating the strong differentiation capacity
of the method.[25]
Given the different drug delivery conditions associated with different diseases, the
design of nano/micro medicines capable of responding to these different conditions
is essential, such as pH-responsive, temperature-sensitive, and heat-sensitive micro/nanomedicines.
Appropriate in vitro release conditions are fundamental to confirming effective drug release in the presence
of a triggering stimulus. To achieve an accurate simulation of the in vivo targeted release environment, attention should also be paid to examining key factors
that influence the above behaviors such as temperature, pH, adipose tissue distribution,
metabolism, diffusion barriers (e.g., body fluid viscosity and connective tissues),
enzymolysis, proteins, vascular distribution, drug volume, osmolality, and inflammation.
For heat-sensitive micro/nanomedicines, drug release can be controlled using a thermoresponsive
system that should maintain the drug load at the body temperature (37°C) and deliver
the drug upon moderate local heating (40–42°C), as occurs in some tumors.[26] Sample and isolation methods can be used to evaluate drug release from carriers
that utilize thermoresponsive release mechanisms, since in any case they are not directly
exposed to the temperature of the release medium. However, in dialysis-based methods,
it is important to ensure the integrity of the semipermeable membrane throughout the
temperature range to effectively study drug release from thermoresponsive carriers.
Particle Size and Distribution
The particle size and distribution are the key determinants of carrier-based micro/nanomedicines
as they are important to stability, release/dissolution behavior, pharmacokinetics,
and biodistribution. Therefore, the Guidelines for Microparticle Formulations in the
Chinese Pharmacopeia require data or graphs of the mean value of the particle size
and its distribution.
Particle size determination is mainly divided into nonimaging and imaging methods.
Dynamic light scattering (DLS) is the most classic nonimaging method. The DLS's average
particle sizes are classified into volume, intensity, and number distributions according
to the algorithms, presenting different values. To avoid ambiguity, the guidelines
issued by the FDA and the CDE adopt the volume and mass distribution pattern.[27] DLS has drawbacks in that large particles may block the scattered light signals
of small particles. If the particles are prone to aggregation and sedimentation, the
accuracy of the method is impaired. Carrier-based micro/nanomedicines that are incompatible
with DLS can be characterized using imaging techniques such as transmission electron
microscopy (TEM) and scanning electron microscopy (SEM). In addition to the particle
size, information such as the morphology and degree of aggregation can be obtained.
Particle size distribution is commonly calculated as a number or volume/mass distribution.
FDA recommends polydispersity index (PDI) to evaluate the size distribution, which
is the ratio of mass average particle size by number average particle size.[28] For micro- and nanomedicines such as liposomes, PDI values of less than 0.5 are
usually required. The smaller the PDI value, the more homogeneous the size distribution.
Generally, the system is considered very homogeneous at a PDI value below 0.1. Span
is another indicator, being calculated by (D90–D10)/D50. D10, D50, and D90 mean that
10, 50, and 90% of the total particles are smaller than this size, respectively. A
smaller span represents a narrower distribution. In some cases, the above methods
may not detect large particles in the preparation. The CDE suggests a drug recovery
assay following filtration through a 0.22-μm membrane to evaluate the presence of
large particles.[29]
However, when the particle size distribution shows complex distribution patterns such
as multiple peaks, the traditional examination parameters such as D50 and Span are
no longer applicable. The Earth Mover's Distance (EMD) is a new metric for assessing
the differences between distributions. EMD defines the distance between two histograms
as a transport problem that can be used to measure the difference between two similar
distributions. Therefore, it can be applied to graphical similarity comparisons of
particle size distributions. It provides a data analysis basis for further evaluation
of particle size bioequivalence. CDE calls for a comparison of paclitaxel albumin
nanoparticle generics with reference using population bioequivalence analysis methods
for particle size and particle size distribution. Three batches of each of the generic
drug and the reference are selected, with no less than 10 bottles in each batch and
no less than three parallel determinations for each bottle.
In 1994, the FDA announced that there were two cases of deaths caused by injections
of fat emulsion for parenteral nutrition. The organic calcium and inorganic phosphoric
acid particles added were too large and blocked the blood vessels of the human body,
which eventually led to the death of the patients. Since the diameter of human microvessels
is 4 to 9 μm, FDA requires an evaluation of the percentage of the oil phase volume
occupied by fat globules larger than 5 μm (PFAT5) should not exceed 0.05%, based on
photoresist measurements.
Morphology
The morphology may affect the interaction of carrier-based micro/nanomedicines with
proteins and cell membranes, the drug release, the degradation, and the translocation.
For example, rough surfaces are prone to adsorb drug molecules but often lead to highly
abrupt release.[24] Optical microscopy, SEM, TEM, and atomic force microscopy (AFM) have been used to
observe the morphology of carrier-based micro/nanomedicines. TEM provides better composition
and topography, allowing for atomic-scale resolution. SEM uses an electron beam to
image the surface of the sample, which means that the sample must be electrically
conductive before it can be analyzed. SEM allows the observation of polymer aggregation
and morphology. AFM can be operated under a variety of conditions including air, liquid,
and vacuum. The FDA even requires cryo-electron microscopy (cryo-TEM) for precise
imaging of nanoemulsions and liposomes.[30] For example, cryo-TEM gave a clear visualization of the “honeycomb” structure of
bupivacaine multivesicular liposomes.[30]
Notably, AFM is also a promising strategy for exploring the morphological changes
associated with environmentally responsive conditions for micro/nanomedicines. For
example, Li et al used AFM to investigate the depolymerization behavior of redox-sensitive
polymeric micelles used to deliver the antitumor drug paclitaxel.[31] The micelles were incubated with 10 μmol/L, and 10 and 20 mmol/L glutathione (GSH)
for 24 hours and observed for changes in micelle morphology, replicating the human
plasma GSH concentration (10 μmol/L) and the tumor environment (10 and 20 mmol/L),
respectively. Compared with untreated micelles, micelles treated with 10 μmol/L GSH
showed no significant morphological changes. In contrast, micelles incubated with
10 and 20 mmol/L GSH displayed irregular shapes and increased particle sizes due to
increased aggregation caused by micelle breakage under reducing conditions.
Surface Properties
The surface charge of carrier-based micro/nanomedicines affects their stability, interaction
with cells, and biodistribution. The surface charge is mainly indicated by zeta potential.
The greater the zeta potential is, the stronger repulsion may appear among particles,
reducing the possibility of flocculation, aggregation, and deposition. Generally,
an absolute value greater than 15 mV meets the stability requirements.[32] The measurement methods include phase analysis light scattering, electrophoretic
light scattering (ELS), and tunable resistive pulse sensing.[33] The most established method is ELS. ELS utilizes the Doppler shift of the scattered
light produced by the electrophoretic motion of the particles. The raw optical signal
is analyzed to obtain information about the velocity of the particles, i.e., the movement
of charged particles in the presence of an electric field. The speed of motion (called
the electrophoretic velocity) is directly proportional to the zeta potential. From
this, the zeta potential can be calculated. The zeta potential depends on the measurement
conditions, such as the dispersion medium, ion concentration, pH, and instrument parameters.[34]
Encapsulation Efficiency and Drug Loading
The encapsulation efficiency is the weight ratio of the encapsulated to the total
drug in the preparation, whereas the drug loading is the weight ratio of the loaded
drug to the micro/nanoparticles (the total weight of the loaded drug and the carrier).
The key to determining the encapsulation efficiency/loading is to separate the free
drug from the encapsulated one. The separation methods include size exclusion chromatography,
ultracentrifugation, and ultrafiltration.[35]
Ultracentrifugation exploits the difference in gravity between the free drug and the
intact drug for separation. Ong et al described a two-step process to separate griseofulvin-loaded
liposomes from free griseofulvin.[36] Initially, they centrifuged the suspension at 12,800 × g for 5 minutes to remove undissolved griseofulvin. Then, they ultracentrifuged the
supernatant at 215,000 × g for 2 hours to spin down the griseofulvin-loaded liposome and leave the free dissolved
griseofulvin in the supernatant. The pellet from the first step (containing the free
undissolved griseofulvin) and supernatant from the second step (containing free dissolved
griseofulvin) were combined to quantify the total amount of free griseofulvin. This
method is easy to operate and suitable for more robust carriers such as solid lipid
micro/nanoparticles, polymer micro/nanoparticles, and so on.
Ultrafiltration centrifugation is performed by placing the micro/ nano drug into an
ultrafiltration tube equipped with an ultrafiltration membrane. By centrifugation
at a suitable speed, the free drug can pass through the ultrafiltration membrane under
centrifugal force, while the intact drug is retained. The separation of the two is
thus achieved. However, the existence of “concentration polarization” limits the application
of ultrafiltration.[37] Concentration polarization is due to the fact that in the ultrafiltration process,
solvents and small molecules can pass through the ultrafiltration membrane, while
macromolecules are retained within the membrane, which leads to an increase in the
concentration of macromolecules on the surface of the ultrafiltration membrane, causing
an increase in the osmotic pressure near the membrane, preventing the solution from
continuing to diffuse in the direction of the membrane, and thus decreasing the membrane
permeability of solvents and small molecules.
However, for fragile carriers such as liposomes, micelles, and micro- and nanoemulsions,
high-speed centrifugation will destroy the structure of liposomes, resulting in drug
leakage and low encapsulation rate. Mild conditions such as size exclusion chromatography
should be chosen to prevent drug leakage caused by high external force input. The
method is based on the molecular sieve effect and utilizes the difference in retention
capacity of the intact and free drug on the gel column to achieve separation.[38]
Stability
Stability studies of micro/nanomedicines should include the physical, chemical, and
microbiological stability of the drug product in impact factor tests, accelerated
tests, and long-term tests. Factors that may affect the stability of nanomedicines
include degradation of polymer nanoparticles, aggregation of nanoparticles, degradation
of the drug, leakage of the drug within the carrier, and degradation of surface modification
molecules or coating materials. Attention should be paid to, but not limited to, the
following indicators and their changes: particle size and distribution, entrapment
efficiency, particle shape and charge; aggregation of nanoparticles, dispersibility
of nanoparticles; in vitro dissolution, release or leakage rate, i.e., the ratio of the amount of drug that
leaks into the medium after a certain period to the amount of drug that was encapsulated
in the product prior to storage; degradation of excipients and the drug; the content
of the active ingredient; and the focus of the pharmacopeia in the dosage form used.[4]
[9]
Studies have shown that high temperatures, high humidity, intense light, and high
ionic strength can greatly destabilize micro/nanomedicines.[39]
[40] For example, an increase in temperature can significantly increase the particle
size of the micelles, i.e., the micelles undergo expansion. High ionic concentrations
decrease both the thermodynamic and kinetic stability of micelles. The reason is that
the anions and cations present in the solution can neutralize the charge on the surface
of the micelles, weaken the thickness of the electric double layer on the surface,
and weaken the electrostatic repulsion. Therefore, micelles are easy to merge and
flocculate, and destabilize. To ensure stability, they should be stored under low
temperature/lyophilization, anaerobic and light-protected conditions if possible,
with the addition of metal chelating agents such as ethylenediaminetetraacetic acid
or antioxidants such as vitamin E and ascorbyl palmitate.[41]
[42]
Quality Control of Microspheres
Quality Control of Microspheres
Microspheres are long-acting formulations for subcutaneous and intramuscular injection,
which form a drug reservoir at the injection site and the extended release of the
loaded drug maintains a long-acting effect. Microspheres significantly improve patient
compliance by reducing the administration frequency and providing stable blood levels
for months or even a year. The drugs' diffusion capacity and the microsphere matrix's
degradation affect the drug release pattern.[43] Microspheres have attracted considerable research interest, and many microsphere
formulations are available on the market ([Table 2]). However, the preparation of microspheres is complex, requiring stringent quality
control. Except for the Guidelines for Microspheres Formulation of Chinese Pharmacopoeia,
no detailed guidelines for microsphere formulations have been published. There is
a lack of international consensus on developing and evaluating these formulations.
In this section, the quality control methods for microspheres are discussed.
Table 2
Commercially available microsphere formulations
|
Product
|
Manufacturer
|
Active ingredient
|
Indication
|
Year of approval
|
|
Lurpon Depot
|
Takeda Pharmaceuticals
|
Leuprorelin
|
Prostate and breast cancer
|
1995
|
|
Viadur
|
Bayer
|
Leuprorelin
|
Prostate and breast cancer
|
2000
|
|
Decapeptyl
|
Ipsen Pharma Biotech
|
Triptorelin
|
Prostate cancer, endometriosis, uterine fibroids
|
1986
|
|
Suprefact
|
Merck
|
Buserelin
|
Prostate and breast cancer
|
1990
|
|
Zoladex
|
AstraZeneca
|
Goserelin
|
Prostate cancer, breast cancer
|
2015
|
|
Sandotatin
|
Novartis
|
Octreotide
|
Acromegaly; endometriosis, neuroendocrine tumors
|
1997
|
|
Plenaxis
|
Praecis
|
Abarek
|
Prostate cancer
|
2009
|
|
Signifor Lar
|
Novartis
|
Parreotide
|
Gastric secretory carcinoma
|
2014
|
|
Risperdal Consta
|
Johnson & Johnson
|
Risperidone
|
Schizophrenia
|
1997
|
|
Zilretta
|
Flexion
|
Triamcinolone acetonide acetate
|
Knee pain associated with osteoarthritis
|
2017
|
|
Arestin
|
Alora Pharmaceuticals
|
Minocycline
|
Periodontitis
|
2012
|
|
Depo-Provera
|
Pfizer
|
Medroxyprogesterone
|
Contraception
|
1992
|
|
Nutropin
|
Genentech
|
Growth hormone
|
Microplasia
|
1998
|
|
Somatuline
|
Ipsen Pharma Biotech
|
Lanreotide
|
Acromegalia
|
2007
|
|
Bydureon
|
AstraZeneca
|
Exenatide
|
Diabetes mellitus
|
2011
|
|
Vivitrol
|
Alkermes
|
Naltrexone
|
Opioid dependence
|
2006
|
In vitro Release
In the case of microspheres, due to their large size, the released drug can be more
easily isolated from the microcarriers using simple centrifugal forces and relatively
little time compared with the methods used for nanocarriers.[44] These gentle conditions allow the structure of the microcarriers to remain intact.
When assessing the release of drugs from these carriers, parameters can be adjusted,
such as sample volume, release medium, centrifugal force, and time.
Microspheres are long-lasting, slow-release formulations with a drug-release cycle
of up to months or years. Therefore, accelerated release methods need to be developed.
Generally, temperature, pH, surfactants, and organic solvents are adjusted to accelerate
drug release.[45] The method development process should focus on in vivo correlation, adapt to the release mechanism of microspheres, and distinguish the
release pattern from different formulations or preparation processes.[45]
In addition, in vitro release trials of long-acting injectables without acceleration take a long time,
during which the stability of the drug in the release medium must be taken into account.[46] However, the amount of drug degraded in the release medium cannot be directly determined
by conventional physical or chemical methods of quantitative analysis. The existing
API degradation ratio method assumes that the drug released from the formulation degrades
at the same rate as the pure API when directly placed in the same release medium.
The API solution is employed in conjunction with the release degree test method. The
release percentage is calculated as the ratio of the drug concentration from the formulation
to that of the API in their respective release media at each sampling time point.
The degradation behavior of API in solution differs from that in the formulation during
release studies. In an API solution, the drug is immediately available for degradation
upon contact with the dissolution media. In contrast, the API in an encapsulated formulation
must first be released from its carrier into the free state before interacting with
the media and undergoing degradation. This delayed release process explains why experimental
data and theoretical models demonstrate that the raw material degradation ratio method
overestimates the release results compared with the actual drug formulation. To solve
the above problem, the double-zero model method is now developed.[47] The key is to prepare the release medium solution of the active ingredient of the
drug in the same method to determine and calculate the degradation rate of the active
ingredient of the drug. This method solves the problem of overestimated results in
the API degradation ratio method.
The release of microspheres mainly consists of an early burst release phase, a delayed
release phase, and a rapid release phase.[48] The drugs adsorbed on the microsphere surfaces diffuse rapidly into the medium,
leading to a burst release effect. This will cause a rapid loss of the loaded drugs
in a short period and shorten the duration of therapeutic efficacy, limiting the wide
application of microspheres. It is necessary to adopt the burst release rate as an
indicator in the quality control process. The burst effect is related to the particle
size of the microspheres, the nature of the drug, the properties of the polymer, the
porosity, and the drying method.[49] Burst release experiments are usually performed using the experimental conditions
of normal release. In the current quality standard for peptide microspheres for injection
with a dosing cycle of 1 month, the sudden release experiment is conducted at 37°C,
using phosphate buffer pH 7.4 as the release medium, and the release within 24 hours
is determined to be no more than 3%, and the release within 24 hours in acetate buffer
pH 4.0 is no more than 5%. Guidelines for Microsphere Formulation of Chinese Pharmacopoeia
stipulate that the burst release should be less than 40% within 0.5 hours.
A drug release curve composed of at least three points is required to characterize
the different release phases of microspheres accurately. The three sampling points
should embody the burst release, the drug-release phase, and the release level, respectively.
The “Guidelines for Slow-release, Controlled-release, and Delayed-release Formulations”
stipulate that the duration of the whole release process should not be less than the
administration interval, and the cumulative release should be more than 90%. The release
is affected by the type and concentration of the polymer, the solubility and stability
of the drug in the release medium, and the affinity of the drug to the microspheres.[50] The release methods for microspheres included in the FDA database are summarized
in [Table 3].
Table 3
The release methods for microspheres included in the Food and Drug Administration
database
|
Product
|
USP apparatus
|
Speed
|
Medium
|
Sampling times
|
|
Trelstar
|
USP II (Paddle)
|
200
|
50 mL methanol and 950 mL water
|
1, 8, 24, 96, and 168 h
|
|
Zilretta
|
USPII (Paddle)
|
75
|
10 mmol/L phosphate buffer (pH 7.2) containing 0.3% SDS and 0.02% sodium azide (1,000 mL,
35°C)
|
1, 2, 4, 8, 12, 16, 24, 36, 48, 72, 96, and 120 h
|
|
Vivitrol
|
USP IV (Flow-Through Cell)
|
–
|
Phosphate buffered saline with 0.02% Tween 20 and 0.02% sodium azide, pH 7.4 (final
osmolality should be 270 ± 20 mOsm) at 37°C
|
–
|
|
Risperidone
|
USP IV (Flow-Through Cell)
|
–
|
–
|
–
|
|
Sandostatin LAR Depot
|
USP IV (Flow-Through Cell)
|
–
|
–
|
–
|
|
Paliperidone Palmitate
|
USP II (Paddle)
|
50
|
0.489% (w/v) polysorbate 20 in 0.001 N HCl 25°C
|
1.5, 5, 8, 10, 15, 20, 30, and 45 min
|
Microspheres are mostly long-lasting and slow-release formulations, and as a result,
effective accelerated in vitro release methods have become one of the key points for the in vitro release of microspheres. Generally, on the basis of sudden-release and normal-release
experiments, the release of the drug from the microspheres is accelerated by adjusting
the conditions of temperature, pH, surfactants, and organic solvents. Currently, researchers
are using a modified flow cell method to perform in vitro release assays of Risperdal Consta under real-time and accelerated testing conditions.[51] A 12-mm diameter flow cell was filled with glass beads of 1-mm particle size and
250 mL of 0.05 mol/L phosphate-buffered saline solution at pH 7.4 containing 0.1%
sodium azide was added. This was adjusted so that it flowed through the flow cell
(equipped with a 0.45-μm regenerated cellulose filter membrane) at a flow rate of
8 mL/min. The temperature was maintained at 37 ± 0.1°C and 45 ± 0.1°C for real-time
and accelerated tests, respectively, which demonstrated an initial burst (24-hour
release) of 1.6%, followed by a release lag of about 24 days, with a rapid release
of the drug from day 24 to day 40. Increasing the temperature from 37 to 45°C resulted
in a breakthrough (24-hour release) of approximately 2%, with a lag time of 4 days
instead of 24 days at 37°C, followed by a rapid drug release period of approximately
7 days. At 50 and 54.5°C, the total release time is further reduced to approximately
3 and 2 days, respectively. Accelerated in vitro release profiles obtained at 50 and 54.5°C showed a linear correlation between the
in vitro–in vivo profiles after time scaling.[52] Accelerated release can also be achieved by adding organic solvents and surfactants
to the release medium. Organic solvents can increase the total porosity of microspheres
to achieve accelerated release.[29] In the current quality standard of peptide microsphere injection, the temperature
of the accelerated test is 37, 48, 50, and 61°C; the pH value is 2.0, 7.0, and 10.0;
and the surfactants added are polyvinyl alcohol, polysorbate 80, polysorbate 20, and
sodium dodecyl sulfate. The accelerated experimental end times were 24, 30, 48, and
72 hours, which were generally controlled within 10% of the time of the normal-release
experiments, with individual preparations at 21 and 50%.
Quality Control of Excipients
The molecular weight and distribution of the polymer affect the burst release and
release rate of microspheres. A wider molecular weight distribution is associated
with faster hydrolysis and degradation of the microspheres, leading to faster drug
release. Therefore, it is important to control the molecular weight and distribution,
which is now commonly determined by gel permeation chromatography.[53]
Particle Size and Distribution
Particle size and distribution are important factors that affect the release and circulation
time of the microspheres in the body.[54] Generally, the small-sized microspheres present a high burst release due to the
large surface area. Microspheres with large particle sizes are easily recognized by
macrophages and removed from the body.[55] The particle size also affects the syringe's capacity. The inner diameter of the
needle ranges from 0.34 to 0.84 mm for intramuscular injection, while a range of 0.26
to 0.34 mm for subcutaneous injection.
Shape and Morphology
The shape and morphology of microspheres may affect the drug loading and release.
Microspheres with rough surfaces and pores exhibit severe burst release.[56] The microspheres prepared by emulsification have porous structures due to the migration
and replacement of organic solvent from the inside of the microspheres to the outside.
The drug payloads migrate with the organic solvent to the microsphere surface during
the drying process, exacerbating the burst release. The ideal microsphere morphology
should be round and smooth.
Quality Control of Micelles
Quality Control of Micelles
Micelles can enhance the solubility of the co-formulated drugs and improve their pharmacokinetic
and biodistribution, thus reducing their tissue toxicity and significantly improving
their therapeutic index. Currently, several polymeric micellar formulations, such
as Nanoxel M, NK105, NC-6004, SP1049C, and NC-4016, are in clinical development, while
paclitaxel micelles, Genexol-PM, has been first approved in Korea.
In vitro Release
The in vitro release for micelles encapsulating hydrophobic drugs is generally evaluated by the
dialysis method. To determine the release profile of Genexol-PM, Werner et al aliquoted
0.1 mL of Genexol-PM solution into Slide-A-Lyzer MINI dialysis microtubes (Pierce,
Rockford, Illinois, United States) with a molecular weight cutoff of 10 kDa.[57] Dialysis was performed at 37°C in 4 L of phosphate buffer solution, and samples
were taken periodically for measurement. The results showed the slow release of paclitaxel
from the micelles under sink conditions, with 65% release at 24 hours and 95% release
at 48 hours. Centrifugation-based in vitro release is recommended for micelles encapsulating hydrophilic drugs or proteins.[58]
For environmentally responsive micelles, specific in vitro release methods are required. Drug release from photosensitive micelles can be assessed
by dialysis by keeping the sample under irradiation during the experiment.[59] Gao et al prepared micelles modified with alginate for cancer tissue targeting and
assessed drug release in the presence of α-l-focusing enzyme (AFU),[60] a lysosomal enzyme that is overexpressed in some tumors such as hepatocellular carcinoma.
This enzyme is a trigger for micelle breakdown, and as a result, drug release was
significantly enhanced in the presence of AFU.
Critical Micelle Concentration
The minimum concentration at which amphiphiles form micelles is called the critical
micelle concentration (CMC). This parameter provides crucial information about the
stability and formation conditions of micelles. In addition, it can guide the selection
of appropriate amphiphile concentrations to ensure efficient drug encapsulation and
controlled release. The commonly used measurement methods include the surface tension
method and the pyrene fluorescence method. The surface tension of the amphiphile solution
shows a break at the CMC, which remains virtually constant with a further increase
in concentration. The concentration at the breakpoint represents the CMC of the amphiphiles.
Upon excitation at 335 nm, five electronic vibrational peaks appear in the fluorescence
emission spectrum of pyrene. The ratio of the first and third of these vibrational
peaks (I1
/I3
) strongly depends on the polarity of the environment in which the probe is located.
At lower than CMC, micelles have not yet formed and at this time pyrene is present
in the aqueous phase. When the polymer concentration is higher than CMC, micelles
are formed and the probe is solubilized into the micellar core due to its hydrophobicity.
Due to the large difference in polarity between the two environments, there is a significant
change in I1
/I3
, and the CMC value can be obtained by plotting the I1
/I3
versus concentration curve and using the tangent method.
The aggregation-caused quenching (ACQ) fluorophore is a novel and sensitive probe
for the determination of CMC.[61] Bearing the hydrophobicity, planarity, and rigidity of fluorophores with an aza-BODIPY
framework, ACQ probes emit fluorescence in molecular dispersion but are quenched in
water due to aggregation. Therefore, the environmental change before and after the
formation of micelles leads to the fluorescence switching of the ACQ probes from quenched
to illuminated.[61] The emergence of the ACQ fluorescence indicates the CMC of the amphiphiles.[62]
[63]
Quality Control of Liposomes
Quality Control of Liposomes
Liposomes offer the advantages of good biocompatibility, low toxicity, and immunogenicity,
and the ability to load hydrophilic and hydrophobic drugs. Hence, it has attracted
much attention and is employed in various therapeutic areas, such as antitumor, antifungal,
anesthesia and analgesia, photodynamic therapy, vaccines, and nucleic acid delivery
([Table 4]).
Table 4
Commercially available liposomal drug products
|
Product
|
Manufacturer
|
Active ingredient
|
Indication
|
Year of approval
|
|
Doxil
|
Sequs
|
Doxorubicin hydrochloride
|
Ovarian cancer, HIV-related Kaposi's sarcoma, myeloid melanoma
|
1995
|
|
Myocet
|
Elan
|
2000
|
|
DaunoXom
|
NeXstar
|
Daunorubicin citrate
|
Acute myeloid leukemia
|
1996
|
|
Depocyt
|
Pacira
|
Cytosine arabinoside
|
Neoplastic meningitis
|
1999
|
|
Marqibo
|
Talon
|
Vincristine
|
Philadelphia chromosome-negative acute lymphoblastic leukemia
|
2009
|
|
Mepact
|
Takeda
|
Mifamurtide
|
Osteosarcoma
|
2009
|
|
Onivyde
|
Merrimack
|
Irinotecan
|
Metastatic adenocarcinoma
|
2015
|
|
Vyxeos
|
Jazz
|
Adriamycin, cytarabine
|
Acute myeloid leukemia
|
2017
|
|
Abelcet
|
Enzon
|
Amphotericin B
|
Fungal infection
|
1995
|
|
AmBisome
|
NeXstar
|
Amphotericin B
|
Cryptococcal meningitis in AIDS patients
|
2000
|
|
Arikayce
|
Amikacin
|
Amikacin
|
Mycobacterium avium complex lung disease
|
2018
|
|
DepoDur
|
Pacira
|
Morphine sulfate
|
Analgesic anesthesia
|
2004
|
|
Exparel
|
Pacira
|
Bupivacaine
|
Analgesic anesthesia
|
2011
|
|
Visudyne
|
Novartis
|
Verteporfin
|
Choroidal neovascularization under the fovea of the macula
|
2000
|
|
Epaxal
|
Crucell Berna
|
Hepatitis A vaccine
|
Hepatitis A
|
1993
|
|
Inflexal V
|
Crucell Berna
|
Influenza vaccine
|
Influenza
|
1997
|
|
Mosquirix
|
GSK
|
Malaria vaccine
|
Malaria
|
2015
|
|
Shingrix
|
GSK
|
Herpes zoster vaccine
|
Herpes zoster
|
2017
|
In vitro Release/Leakage
The in vitro release/leakage rate is one of the most critical quality controls for liposomes,
which is closely related to the safety and efficacy of the drug in vivo. For in vitro testing, physiological media (e.g., the simulated medium or human plasma) and appropriate
agitation should be adopted to mimic the in vivo environment.[28]
[29]
[34]
[64]
[65] This in vitro test aims to predict whether the drug remains stable in circulation to avoid premature
leakage and whether rapid release can be achieved at the target. [Table 5] shows the conditions recommended by the CDE for the examination of doxorubicin hydrochloride
liposomes.[66]
Table 5
The in vitro leakage/release conditions recommended by the Center for Drug Evaluation for doxorubicin
hydrochloride liposomes
|
Conditions
|
Aim
|
The rationale
|
|
24 h at 37°C in 50% human plasma
|
Evaluation of the stability of liposomes in the circulation
|
Plasma can simulate blood conditions
|
|
24 h at 37°C in pH 5.5, 6.5, and 7.5 buffers
|
Simulation of drug release in normal tissue, around cancer cells, or within cancer
cells
|
Normal tissue: pH 7.3; cancer tissue: pH 6.6; endosomes and lysosomes: pH 5–6
|
|
12 h in pH 6.5 buffer over a range of temperatures (43, 47, 52, 57°C) or until fully
released
|
Evaluation of lipid membrane integrity
|
The phase transition temperature of a lipid membrane is determined by the nature of
the lipid bilayer (e.g., rigidity, stiffness, and chemical composition). Differences
in drug release at different temperatures reflect subtle differences in the nature
of the lipid bilayer
|
|
Sonication at low frequency (20 kHz) at 37°C for 2 h or until fully released
|
Evaluation of the status of encapsulated drugs
|
Low-frequency ultrasound (20 kHz) disrupts the lipid bilayer by briefly introducing
a pore-like defect, allowing doxorubicin sulfate precipitation in liposomes to dissolve
and release doxorubicin
|
For the separation of the encapsulated drug from the free drug in the release medium,
new methods such as dispersion releaser technology can be used.[67] The unit can be used in conjunction with the USP Instrument 1/2 in standard or mini-container
configurations. The dialysis tubing is mounted around the housing of the donor chamber,
which is continuously agitated by a paddle stirrer. Propulsive force is transferred
to a magnetic stir bar in the recipient chamber. The entire assembly is attached to
the motor of the dissolution tester. Samples are collected from the receiving chamber
at defined time points. In addition, the system collects the sample from the donor
chamber. The diffusion of the dialysis membrane is supported by a directed fluid flow.
They are more selective and sensitive and faster than the traditional methods.[23] Currently, the determination of in vitro release of the marketed injectable liposome Foscan as well as the self-developed
liposome Foslip in simulated physiological media has been achieved using the dispersive
release technique.[68]
[69]
During circulation, the leaked/released drug from the formulation binds to and further
induces leakage/release from blood cells in the bloodstream as well as cellular phospholipid
bilayer membranes in various tissues. To more closely resemble the physiological environment,
researchers have developed a donor–acceptor type in vitro drug release method to mimic the process of the drug from the formulation to the
phospholipid cell membrane.[70] Drug-carrying large unilamellar vesicles of approximately 100 nm in diameter were
used as donors and coincubated with a large number of multilamellar vesicles acting
as acceptors (molar ratio 1/100), and the multilamellar vesicles were utilized to
mimic the phospholipid membranes, which in turn led to the calculation of the leakage
rate of the drug in vivo. This method specifically predicts the in vivo leakage/release characteristics of liposomal drugs compared with the traditional
dialysis bag method using plasma as a medium and has been applied to study the correlation
between in vitro data and in vivo release of doxorubicin, verapamil, and ceramide liposomes.[71]
Quality Control of Active Pharmaceutical Ingredient and Excipients
Liposomes comprise APIs, and structural and functional excipients, including phospholipids,
cholesterol, PEGylated phospholipids, and nonlipids. The composition determines liposomes'
structure, stability, and performance.[34]
[65] Therefore, CDE and FDA require the characterization, the identification, the impurity
control, the content determination, and the stability examination of the structural
and functional components, as well as the evaluation of the thermodynamic properties
of the lipid membranes.[34]
[65]
For example, in the case of doxorubicin hydrochloride liposomes, the content of each
component, such as cholesterol (limit 2.87–3.51 mg/mL), hydro phosphatidylcholine
(limit 2.87–3.51 mg/mL), PEG2000-DSPE (limit 8.62–10.54 mg/mL), the ratio of the content
of hydro phosphatidylcholine to PEG2000-DSPE (limit 0.28–0.38), sulfate and PEG2000-DSPE
(limit 0.28–0.38), should be determined. In addition, lipid impurities must be monitored
and controlled, such as trans and free fatty acids (limit of 0.5 mg/mL), hydrolysis
products like lysophosphatidic acid and hydrogenated lysophosphatidylcholine (limit
of 0.5 mg/mL), and oxidative degradation products like unsaturated fatty acids. The
solvents and catalysts used in the synthesis or purification process should also be
controlled. These impurities affect the stability of the formulation, leading to a
decrease in the rigidity of the lipid bilayer and the leakage of the APIs. Parameters
such as solubility, nonpolar lipids, peroxide, iodine, moisture, and microbiological
limits must be controlled for liposomes containing hydrogenated soy phosphatidylcholine.
Solubility, solution clarity and color, microbiological limits, and content should
be regulated for pegylated phosphatidylethanolamine.
Particle Size and Distribution
CDE and MHLW (Ministry of Health, Labor and Welfare of Japan) recommend the use of
DLS or laser diffraction to determine the liposome particle size.[34]
[64] Particular attention should be given to information on the measurement conditions,
the distribution pattern of the particle size, and, if necessary, the basis for the
choice. A volume or mass distribution-based size is recommended, while the size distribution
should be expressed as a PDI.[64] The particle size is 20 to 80 nm for small unicompartmental liposomes, 0.1 to 1
μm for large unicompartmental liposomes, and 1 to 5 μm for multicompartmental liposomes.
Zeta Potential
Zeta potential affects liposome aggregation, in vivo clearance, tissue distribution, and interaction with cells. The zeta potential is
mainly determined by ELS. It converts zeta potential into charged particle flux measurements
by electrochemical principles and uses the Doppler effect of incident light waves
to measure this flux. The solvent used, pH, and conductivity are important influencing
factors. Doxorubicin hydrochloride liposomes are required to have a potential between
−16 and −11 mV.[34]
[66]
The State and Spatial Distribution of the Loaded Active Pharmaceutical Ingredient
The state and spatial distribution of the loaded drug have a great impact on the stability
and release pattern of liposomes. For example, Doxil is prepared by an active loading
technique, leading to precipitation of doxorubicin inside the liposomes. The precipitated
state effectively prevents the early leakage of the drug.[72]
[73] CDE recommends using electron microscopy and X-ray diffraction to evaluate the state
of doxorubicin inside liposomes.[66] The location of the drug being loaded in liposomes, such as in the internal aqueous
phase, in the middle of the lipid bilayer, and on the surface of liposomes, affects
the drug's release/leakage and needs to be investigated.[74]
Stability
Liposomes are easy to form an aggregation and cause drug leakage in vitro. The in vivo degradation and elimination of liposomes reduce its delivery capacity. Stability
studies should elucidate the physical, chemical, and microbial stability of liposomes.
Due to their poor physical stability, liposomes are prone to fusion and aggregation
during storage. The instability of phospholipid membranes, changes in particle size,
and zeta potential can cause instability in the physical form and structure of liposomes.
For Doxil, the following properties should be tested in accelerated and long-term
tests to determine the physical stability: particle size, zeta potential, morphology,
turbidity, encapsulation rate, in vitro release, and leakage.
The chemical stability of liposomes is inferior because lipids, the main component
of liposomes, are easily hydrolyzed to form lysophospholipids and free fatty acids
or oxidized. The phospholipid hydrolysis of liposomes is a spontaneous process. With
the increase of free fatty acid, the pH value of the system decreased. This further
promoted the hydrolysis of phospholipids, resulting in the leakage of liposomes encapsulating
drugs and the production of toxic hydrolysates such as lysophosphatidylcholine. Hydrolysis
of lecithin, saturated soybean phospholipids, and phosphatidylglycerides is affected
by pH. These phospholipid components are most stable at pH 6.5 and have the smallest
hydrolysis rate constants. Therefore, a buffer solution was added to the liposome
suspension to keep the pH in the most stable range for liposomes. Liposomes are mostly
composed of phospholipids with unsaturated bonds that are highly oxidized. Oxidation
can decrease the fluidity of the liposome membrane and increase the leakage of drugs.
Moreover, products such as peroxides and malondialdehyde produced by the oxidation
of phospholipids are toxic to the human body. Oxidative degradation can be reduced
by the use of high-quality raw materials, by the addition of antioxidants, and by
the preparation of liposomes under an oxygen-free atmosphere. Storage at low temperatures
reduces the rate of oxidation as well. Furthermore, the use of partially saturated
phospholipids could be a better choice than the phospholipids carrying polyunsaturated
fatty acyl chains (natural egg phosphatidylcholine). In contrast to oxidative degradation,
hydrolysis of phospholipids can only be completely prohibited by the removal of water
by (freeze) drying. However, due to the physical stability problems encountered with
freeze-drying of liposomes containing hydrophilic, nonbilayer interacting, low molecular
weight drugs, e.g., loss of drug after rehydration and tendency to increase in mean
particle size, storage of liposomes as aqueous dispersions may be preferred. In aqueous
dispersions, storage temperature, and pH are the two main parameters that affect phospholipid
hydrolysis. For long-term stability, it is recommended to store liposomes at low temperatures
(4–6°C in a refrigerator) and to adjust the pH of the dispersion to a near-neutral
pH (pH 6.5)—when phospholipids have maximum stability.
Conclusion
Quality control of carrier-based micro- and nanomedicines covers the basic characteristics
such as content, related substances, and nano-specific characteristics including particle
size, morphology, in vitro release, surface properties, and entrapment efficiency. This ensures the stability
and effectiveness of the drug formulation. Guidelines related to quality control of
micro/nanomedicines need to be supplemented with more predictive, scientific, and
standardized techniques. To better reflect in vivo release behavior and to reduce the failure rate of clinical trials, the development
process should focus on more standardized and easier-to-operate commercial dialysis
devices such as Float-A-Lyzer, Side-Bi-Side, and dispersion releaser technology. Guidelines
for the development of ex vivo and in vivo correlation models should be initiated, focusing on in vitro release assessment devices such as GastroPlus, two-stage antidialysis, subcutaneous
injection site simulator, and donor–recipient models. For different types of drug
delivery systems, more specific quality standards should be developed based on administration
purposes, drug release mechanisms, preparation methods, and raw and auxiliary materials.
International cooperation and exchanges must be strengthened to promote the translational
and clinical studies of carrier-based micro- and nanomedicines.
