The Quality Control of Micro- and Nanomedicines: Compliance with Regulatory Guidelines

Abstract

Carrier-based micro- and nanomedicines, such as microspheres, liposomes, and micelles, offer enhanced benefits over traditional medications, including improved bioavailability, targeted delivery, and reduced toxicity. Unlike conventional drugs, the particle size, shape, surface charge, and surface chemical properties of carrier-based micro/nanomedicines are key factors in determining their efficacy and toxicity. Even minor deviations in their preparation can significantly impact their in vivo performance. Therefore, the quality control of carrier-based micro/nanomedicines is more complicated than for conventional drugs. Regulatory authorities from the United States and China have established guidelines for their quality assessment. These guidelines categorize quality indicators into basic characteristics such as pH, viscosity, microbial limits, and nano-specific characteristics, including particle size, morphology, and stability. This review summarizes the significance and methodologies for evaluating these characteristics based on pharmacopeias and guidelines, emphasizing the distinct quality control requirements for different types of carrier-based micro- and nanomedicines.

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

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

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

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

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.

Conflict of Interest

None declared.

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Publication History

Received: 24 November 2024

Accepted: 01 April 2025

Article published online:
21 May 2025