Special Issue "Advances in Characterization Methods for Drug Delivery Systems"

A special issue of Pharmaceutics (ISSN 1999-4923). This special issue belongs to the section "Drug Delivery and Controlled Release".

Deadline for manuscript submissions: closed (31 July 2022) | Viewed by 29201

Special Issue Editors

Department of Chemistry “Ugo Schiff”, Center for Colloids and Surface Science (CSGI), University of Florence, Via della Lastruccia 3, 50019 Sesto Fiorentino, Italy
Interests: physical chemistry; soft matter; biotechnology small angle scattering
Department of Pharmacy, Università degli Studi di Napoli Federico II, via Domenico Montesano 49, 80131 Naples, Italy
Interests: drug delivery; nanomedicine; drug targeting; liposomes; self-assembling nanoparticles; systemic delivery; brain delivery; wound healing
Special Issues, Collections and Topics in MDPI journals
Department of Pharmacy, University of Naples Federico II, Via Domenico Montesano 49, 80131 Naples, Italy
Interests: nanotechnology; lipid nanovectors; skin delivery; nucleic acid delivery
Special Issues, Collections and Topics in MDPI journals

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Assistant Guest Editor
1. Department of Biotechnology, Chemistry and Pharmacy, University of Siena, via Aldo Moro 3, 53100 Siena, Italy
2. Department of Chemistry “Ugo Schiff” & CSGI, University of Florence, via della Lastruccia 3, 50019 Sesto Fiorentino, Italy
Interests: soft matter; lipid nanovectors; drug delivery; structural characterization

Special Issue Information

Dear Colleagues,

Advances in the design of novel delivery strategies have increased opportunities for the development of innovative therapies for acute and chronic diseases. The advent of nanotechnology in the field of pharmaceutics and the development of “smart” biomaterials have overcome drawbacks such as poor biopharmaceutical profile, especially in the case of macromolecules, while ensuring more efficient and targeted delivery of actives molecules. Innovative delivery approaches, i.e., based on colloidal systems, hydrogels, bioconjugates, macromolecular complexes, stimuli-responsive nanocarriers, etc., can be used to address the issues related to drug solubility, chemical-physical and biological instability of the active molecule, disadvantageous pharmacokinetics, and biodistribution. Finally, different platforms have been used to prolong drug release, thus, improving patient compliance.

Undoubtedly, the current landscape that drives research in drug delivery is interdisciplinary, demanding the deepest knowledge of technological, physicochemical, and biological aspects. The fundamental understanding of the properties and functionality of delivery systems requires in-depth, high-resolution characterization, specifically tailored to the system class and its application. The complexity of such formulations often calls for an integrated approach, where solely the combined use of different techniques can grant a full picture. Moreover, while the physicochemical and structural characterization is the first step for rationale carrier design and investigation, the analytical study of the composition, interaction, and release mechanisms, followed by toxicity and biocompatibility assessment, also represent key matters.

This Special Issue will explore current significant technical and methodological advancements in the characterization of innovative drug delivery systems, with a focus on the applications and best-suited approaches for each specific case. The reported classes of methods will include, but will not be limited to the following:

Size characterization (dynamic light-scattering), high-resolution structural techniques (X-ray and neutron scattering, electron, confocal, and fluorescence microscopy); thermodynamic methods (calorimetry, rheology); computational methods; magnetic resonances and other spectroscopic methods; analytical techniques (HPLC, Mass Spectrometry); surface characterization (porosity, Zeta Potential), cytotoxicity (cell culture lines, primary cell culture), hemocompatibility and biocompatibility (in vitro and in vivo studies).

Prof. Dr. Giuseppe De Rosa
Prof. Dr. Sandra Ristori
Dr. Virginia Campani
Ms. Ilaria Clemente
Guest Editors

Manuscript Submission Information

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Submitted manuscripts should not have been published previously, nor be under consideration for publication elsewhere (except conference proceedings papers). All manuscripts are thoroughly refereed through a single-blind peer-review process. A guide for authors and other relevant information for submission of manuscripts is available on the Instructions for Authors page. Pharmaceutics is an international peer-reviewed open access monthly journal published by MDPI.

Please visit the Instructions for Authors page before submitting a manuscript. The Article Processing Charge (APC) for publication in this open access journal is 2900 CHF (Swiss Francs). Submitted papers should be well formatted and use good English. Authors may use MDPI's English editing service prior to publication or during author revisions.

Keywords

  • characterization methods
  • drug delivery systems
  • targeted delivery
  • nanoparticle characterization
  • high-resolution structural techniques
  • microscopy techniques
  • thermodynamic methods
  • cytotoxicity
  • biocompatibility

Published Papers (10 papers)

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Research

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Article
Synthesis, Formulation and Characterization of Immunotherapeutic Glycosylated Dendrimer/cGAMP Complexes for CD206 Targeted Delivery to M2 Macrophages in Cold Tumors
Pharmaceutics 2022, 14(9), 1883; https://doi.org/10.3390/pharmaceutics14091883 - 06 Sep 2022
Cited by 3 | Viewed by 1675
Abstract
Anti-tumor responses can be achieved via the stimulation of the immune system, a therapeutic approach called cancer immunotherapy. Many solid tumor types are characterized by the presence of immune-suppressive tumor-associated macrophage (TAMs) cells within the tumor microenvironment (TME). Moreover, TAM infiltration is strongly [...] Read more.
Anti-tumor responses can be achieved via the stimulation of the immune system, a therapeutic approach called cancer immunotherapy. Many solid tumor types are characterized by the presence of immune-suppressive tumor-associated macrophage (TAMs) cells within the tumor microenvironment (TME). Moreover, TAM infiltration is strongly associated with poor survival in solid cancer patients and hence a low responsiveness to cancer immunotherapy. Therefore, 2′3′ Cyclic GMP-AMP (2′3′ cGAMP) was employed for its ability to shift macrophages from pro-tumoral M2-like macrophages (TAM) to anti-tumoral M1. However, cGAMP transfection within macrophages is limited by the molecule’s negative charge, poor stability and lack of targeting. To circumvent these barriers, we designed nanocarriers based on poly(amidoamine) dendrimers (PAMAM) grafted with D-glucuronic acid (Glu) for M2 mannose-mediated endocytosis. Two carriers were synthesized based on different dendrimers and complexed with cGAMP at different ratios. Orthogonal techniques were employed for synthesis (NMR, ninhydrin, and gravimetry), size (DLS, NTA, and AF4-DLS), charge (DLS and NTA), complexation (HPLC-UV and AF4-UV) and biocompatibility and toxicity (primary cells and hen egg chorioallantoic membrane model) evaluations in order to evaluate the best cGAMP carrier. The best formulation was selected for its low toxicity, biocompatibility, monodispersed distribution, affinity towards CD206 and ability to increase M1 (STAT1 and NOS2) and decrease M2 marker (MRC1) expression in macrophages. Full article
(This article belongs to the Special Issue Advances in Characterization Methods for Drug Delivery Systems)
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Article
Structuring and De-Structuring of Nanovectors from Algal Lipids: Simulated Digestion, Preliminary Antioxidant Capacity and In Vitro Tests
Pharmaceutics 2022, 14(9), 1847; https://doi.org/10.3390/pharmaceutics14091847 - 01 Sep 2022
Cited by 1 | Viewed by 1052
Abstract
Biocompatible nanocarriers can be obtained by lipid extraction from natural sources such as algal biomasses, which accumulate different lipid classes depending on the employed culture media. Lipid aggregates can be distinguished according to supramolecular architecture into lamellar and nonlamellar structures. This distinction is [...] Read more.
Biocompatible nanocarriers can be obtained by lipid extraction from natural sources such as algal biomasses, which accumulate different lipid classes depending on the employed culture media. Lipid aggregates can be distinguished according to supramolecular architecture into lamellar and nonlamellar structures. This distinction is mainly influenced by the lipid class and molecular packing parameter, which determine the possible values of interfacial curvature and thus the supramolecular symmetries that can be obtained. The nanosystems prepared from bio-sources are able to self-assemble into different compartmentalized structures due to their complex composition. They also present the advantage of increased carrier-target biocompatibility and are suitable to encapsulate and vehiculate poorly water-soluble compounds, e.g., natural antioxidants. Their functional properties stem from the interplay of several parameters. Following previous work, here the functionality of two series of structurally distinct lipid nanocarriers, namely liposomes and cubosomes deriving from algal biomasses with different lipid composition, is characterized. In the view of their possible use as pharmaceutical or nutraceutical formulations, both types of nanovectors were loaded with three well-known antioxidants, i.e., curcumin, α-tocopherol and piperine, and their carrier efficacy was compared considering their different structures. Firstly, carrier stability in biorelevant conditions was assessed by simulating a gastrointestinal tract model. Then, by using an integrated chemical and pharmacological approach, the functionality in terms of encapsulation efficiency, cargo bioaccessibility and kinetics of antioxidant capacity by UV-Visible spectroscopy was evaluated. Subsequently, in vitro cytotoxicity and viability tests after administration to model cell lines were performed. As a consequence of this investigation, it is possible to conclude that nanovectors from algal lipids, i.e., cubosomes and liposomes, can be efficient delivery agents for lipophilic antioxidants, being able to preserve and enhance their activity toward different targets while promoting sustained release. Full article
(This article belongs to the Special Issue Advances in Characterization Methods for Drug Delivery Systems)
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Article
Improved Functionality of Integration-Deficient Lentiviral Vectors (IDLVs) by the Inclusion of IS2 Protein Docks
Pharmaceutics 2021, 13(8), 1217; https://doi.org/10.3390/pharmaceutics13081217 - 06 Aug 2021
Cited by 2 | Viewed by 1913
Abstract
Integration-deficient lentiviral vectors (IDLVs) have recently generated increasing interest, not only as a tool for transient gene delivery, but also as a technique for detecting off-target cleavage in gene-editing methodologies which rely on customized endonucleases (ENs). Despite their broad potential applications, the efficacy [...] Read more.
Integration-deficient lentiviral vectors (IDLVs) have recently generated increasing interest, not only as a tool for transient gene delivery, but also as a technique for detecting off-target cleavage in gene-editing methodologies which rely on customized endonucleases (ENs). Despite their broad potential applications, the efficacy of IDLVs has historically been limited by low transgene expression and by the reduced sensitivity to detect low-frequency off-target events. We have previously reported that the incorporation of the chimeric sequence element IS2 into the long terminal repeat (LTR) of IDLVs increases gene expression levels, while also reducing the episome yield inside transduced cells. Our study demonstrates that the effectiveness of IDLVs relies on the balance between two parameters which can be modulated by the inclusion of IS2 sequences. In the present study, we explore new IDLV configurations harboring several elements based on IS2 modifications engineered to mediate more efficient transgene expression without affecting the targeted cell load. Of all the insulators and configurations analysed, the insertion of the IS2 into the 3′LTR produced the best results. After demonstrating a DAPI-low nuclear gene repositioning of IS2-containing episomes, we determined whether, in addition to a positive effect on transcription, the IS2 could improve the capture of IDLVs on double strand breaks (DSBs). Thus, DSBs were randomly generated, using the etoposide or locus-specific CRISPR-Cas9. Our results show that the IS2 element improved the efficacy of IDLV DSB detection. Altogether, our data indicate that the insertion of IS2 into the LTR of IDLVs improved, not only their transgene expression levels, but also their ability to be inserted into existing DSBs. This could have significant implications for the development of an unbiased detection tool for off-target cleavage sites from different specific nucleases. Full article
(This article belongs to the Special Issue Advances in Characterization Methods for Drug Delivery Systems)
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Article
Characterization of Liposomes Using Quantitative Phase Microscopy (QPM)
Pharmaceutics 2021, 13(5), 590; https://doi.org/10.3390/pharmaceutics13050590 - 21 Apr 2021
Cited by 5 | Viewed by 2469
Abstract
The rapid development of nanomedicine and drug delivery systems calls for new and effective characterization techniques that can accurately characterize both the properties and the behavior of nanosystems. Standard methods such as dynamic light scattering (DLS) and fluorescent-based assays present challenges in terms [...] Read more.
The rapid development of nanomedicine and drug delivery systems calls for new and effective characterization techniques that can accurately characterize both the properties and the behavior of nanosystems. Standard methods such as dynamic light scattering (DLS) and fluorescent-based assays present challenges in terms of system’s instability, machine sensitivity, and loss of tracking ability, among others. In this study, we explore some of the downsides of batch-mode analyses and fluorescent labeling, while introducing quantitative phase microscopy (QPM) as a label-free complimentary characterization technique. Liposomes were used as a model nanocarrier for their therapeutic relevance and structural versatility. A successful immobilization of liposomes in a non-dried setup allowed for static imaging conditions in an off-axis phase microscope. Image reconstruction was then performed with a phase-shifting algorithm providing high spatial resolution. Our results show the potential of QPM to localize subdiffraction-limited liposomes, estimate their size, and track their integrity over time. Moreover, QPM full-field-of-view images enable the estimation of a single-particle-based size distribution, providing an alternative to the batch mode approach. QPM thus overcomes some of the drawbacks of the conventional methods, serving as a relevant complimentary technique in the characterization of nanosystems. Full article
(This article belongs to the Special Issue Advances in Characterization Methods for Drug Delivery Systems)
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Article
Characterization of Recombinant Adeno-Associated Viruses (rAAVs) for Gene Therapy Using Orthogonal Techniques
Pharmaceutics 2021, 13(4), 586; https://doi.org/10.3390/pharmaceutics13040586 - 20 Apr 2021
Cited by 22 | Viewed by 4473
Abstract
Viruses are increasingly used as vectors for delivery of genetic material for gene therapy and vaccine applications. Recombinant adeno-associated viruses (rAAVs) are a class of viral vector that is being investigated intensively in the development of gene therapies. To develop efficient rAAV therapies [...] Read more.
Viruses are increasingly used as vectors for delivery of genetic material for gene therapy and vaccine applications. Recombinant adeno-associated viruses (rAAVs) are a class of viral vector that is being investigated intensively in the development of gene therapies. To develop efficient rAAV therapies produced through controlled and economical manufacturing processes, multiple challenges need to be addressed starting from viral capsid design through identification of optimal process and formulation conditions to comprehensive quality control. Addressing these challenges requires fit-for-purpose analytics for extensive characterization of rAAV samples including measurements of capsid or particle titer, percentage of full rAAV particles, particle size, aggregate formation, thermal stability, genome release, and capsid charge, all of which may impact critical quality attributes of the final product. Importantly, there is a need for rapid analytical solutions not relying on the use of dedicated reagents and costly reference standards. In this study, we evaluate the capabilities of dynamic light scattering, multiangle dynamic light scattering, and SEC–MALS for analyses of rAAV5 samples in a broad range of viral concentrations (titers) at different levels of genome loading, sample heterogeneity, and sample conditions. The study shows that DLS and MADLS® can be used to determine the size of full and empty rAAV5 (27 ± 0.3 and 33 ± 0.4 nm, respectively). A linear range for rAAV5 size and titer determination with MADLS was established to be 4.4 × 1011–8.7 × 1013 cp/mL for the nominally full rAAV5 samples and 3.4 × 1011–7 × 1013 cp/mL for the nominally empty rAAV5 samples with 3–8% and 10–37% CV for the full and empty rAAV5 samples, respectively. The structural stability and viral load release were also inferred from a combination of DLS, SEC–MALS, and DSC. The structural characteristics of the rAAV5 start to change from 40 °C onward, with increasing aggregation observed. With this study, we explored and demonstrated the applicability and value of orthogonal and complementary label-free technologies for enhanced serotype-independent characterization of key properties and stability profiles of rAAV5 samples. Full article
(This article belongs to the Special Issue Advances in Characterization Methods for Drug Delivery Systems)
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Article
Preparation and Evaluation of Azelaic Acid Topical Microemulsion Formulation: In Vitro and In Vivo Study
Pharmaceutics 2021, 13(3), 410; https://doi.org/10.3390/pharmaceutics13030410 - 19 Mar 2021
Cited by 15 | Viewed by 3028
Abstract
The aim of this study was to design oil in water (O/W) microemulsion formulations for the topical administration of azelaic acid. The permeability of azelaic acid through rat skin and the anti-inflammatory activities of the formulations were conducted to examine the efficacy of [...] Read more.
The aim of this study was to design oil in water (O/W) microemulsion formulations for the topical administration of azelaic acid. The permeability of azelaic acid through rat skin and the anti-inflammatory activities of the formulations were conducted to examine the efficacy of the designed formulations. Skin irritation and stability tests were also performed. The permeability of azelaic acid was significantly increased by using O/W microemulsions as carriers. The edema index of ear swelling percentage was significantly recovered by the 5% drug-loaded formulation and a 20% commercial product, demonstrating that the experimental formulation possessed comparable effect with the commercial product on the improvement of inflammation. The experimental formulation did not cause significant skin irritation compared to the negative control group. Moreover, the drug-loaded formulation also showed thermodynamic stability and chemical stability after storage for 30 days. In conclusion, the O/W microemulsion was a potential drug delivery carrier for azelaic acid topical application. Full article
(This article belongs to the Special Issue Advances in Characterization Methods for Drug Delivery Systems)
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Review

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Review
Cryogenic Electron Microscopy Methodologies as Analytical Tools for the Study of Self-Assembled Pharmaceutics
Pharmaceutics 2021, 13(7), 1015; https://doi.org/10.3390/pharmaceutics13071015 - 02 Jul 2021
Cited by 4 | Viewed by 2994
Abstract
Many pharmaceutics are aqueous dispersions of small or large molecules, often self-assembled in complexes from a few to hundreds of molecules. In many cases, the dispersing liquid is non-aqueous. Many pharmaceutical preparations are very viscous. The efficacy of those dispersions is in many [...] Read more.
Many pharmaceutics are aqueous dispersions of small or large molecules, often self-assembled in complexes from a few to hundreds of molecules. In many cases, the dispersing liquid is non-aqueous. Many pharmaceutical preparations are very viscous. The efficacy of those dispersions is in many cases a function of the nanostructure of those complexes or aggregates. To study the nanostructure of those systems, one needs electron microscopy, the only way to obtain nanostructural information by recording direct images whose interpretation is not model-dependent. However, these methodologies are complicated by the need to make liquid systems compatible with high vacuum in electron microscopes. There are also issues related to the interaction of the electron beam with the specimen such as micrograph contrast, electron beam radiation damage, and artifacts associated with specimen preparation. In this article, which is focused on the state of the art of imaging self-assembled complexes, we briefly describe cryogenic temperature transmission electron microscopy (cryo-TEM) and cryogenic temperature scanning electron microcopy (cryo-SEM). We present the principles of these methodologies, give examples of their applications as analytical tools for pharmaceutics, and list their limitations and ways to avoid pitfalls in their application. Full article
(This article belongs to the Special Issue Advances in Characterization Methods for Drug Delivery Systems)
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Review
Advanced Static and Dynamic Fluorescence Microscopy Techniques to Investigate Drug Delivery Systems
Pharmaceutics 2021, 13(6), 861; https://doi.org/10.3390/pharmaceutics13060861 - 11 Jun 2021
Cited by 7 | Viewed by 2702
Abstract
In the past decade(s), fluorescence microscopy and laser scanning confocal microscopy (LSCM) have been widely employed to investigate biological and biomimetic systems for pharmaceutical applications, to determine the localization of drugs in tissues or entire organisms or the extent of their cellular uptake [...] Read more.
In the past decade(s), fluorescence microscopy and laser scanning confocal microscopy (LSCM) have been widely employed to investigate biological and biomimetic systems for pharmaceutical applications, to determine the localization of drugs in tissues or entire organisms or the extent of their cellular uptake (in vitro). However, the diffraction limit of light, which limits the resolution to hundreds of nanometers, has for long time restricted the extent and quality of information and insight achievable through these techniques. The advent of super-resolution microscopic techniques, recognized with the 2014 Nobel prize in Chemistry, revolutionized the field thanks to the possibility to achieve nanometric resolution, i.e., the typical scale length of chemical and biological phenomena. Since then, fluorescence microscopy-related techniques have acquired renewed interest for the scientific community, both from the perspective of instrument/techniques development and from the perspective of the advanced scientific applications. In this contribution we will review the application of these techniques to the field of drug delivery, discussing how the latest advancements of static and dynamic methodologies have tremendously expanded the experimental opportunities for the characterization of drug delivery systems and for the understanding of their behaviour in biologically relevant environments. Full article
(This article belongs to the Special Issue Advances in Characterization Methods for Drug Delivery Systems)
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Review
Degradation of Drug Delivery Nanocarriers and Payload Release: A Review of Physical Methods for Tracing Nanocarrier Biological Fate
Pharmaceutics 2021, 13(6), 770; https://doi.org/10.3390/pharmaceutics13060770 - 21 May 2021
Cited by 11 | Viewed by 3347
Abstract
Nanoformulations offer multiple advantages over conventional drug delivery, enhancing solubility, biocompatibility, and bioavailability of drugs. Nanocarriers can be engineered with targeting ligands for reaching specific tissue or cells, thus reducing the side effects of payloads. Following systemic delivery, nanocarriers must deliver encapsulated drugs, [...] Read more.
Nanoformulations offer multiple advantages over conventional drug delivery, enhancing solubility, biocompatibility, and bioavailability of drugs. Nanocarriers can be engineered with targeting ligands for reaching specific tissue or cells, thus reducing the side effects of payloads. Following systemic delivery, nanocarriers must deliver encapsulated drugs, usually through nanocarrier degradation. A premature degradation, or the loss of the nanocarrier coating, may prevent the drug’s delivery to the targeted tissue. Despite their importance, stability and degradation of nanocarriers in biological environments are largely not studied in the literature. Here we review techniques for tracing the fate of nanocarriers, focusing on nanocarrier degradation and drug release both intracellularly and in vivo. Intracellularly, we will discuss different fluorescence techniques: confocal laser scanning microscopy, fluorescence correlation spectroscopy, lifetime imaging, flow cytometry, etc. We also consider confocal Raman microscopy as a label-free technique to trace colocalization of nanocarriers and drugs. In vivo we will consider fluorescence and nuclear imaging for tracing nanocarriers. Positron emission tomography and single-photon emission computed tomography are used for a quantitative assessment of nanocarrier and payload biodistribution. Strategies for dual radiolabelling of the nanocarriers and the payload for tracing carrier degradation, as well as the efficacy of the payload delivery in vivo, are also discussed. Full article
(This article belongs to the Special Issue Advances in Characterization Methods for Drug Delivery Systems)
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Review
Challenges in the Physical Characterization of Lipid Nanoparticles
Pharmaceutics 2021, 13(4), 549; https://doi.org/10.3390/pharmaceutics13040549 - 14 Apr 2021
Cited by 36 | Viewed by 3639
Abstract
Nano-sized drug transporters have become an efficient approach with considerable commercial values. Nanomedicine is not only limited to drug delivery by means of different administration routes, such as intravenous, oral, transdermal, nasal, pulmonary, and more, but also has applications in a multitude of [...] Read more.
Nano-sized drug transporters have become an efficient approach with considerable commercial values. Nanomedicine is not only limited to drug delivery by means of different administration routes, such as intravenous, oral, transdermal, nasal, pulmonary, and more, but also has applications in a multitude of areas, such as a vaccine, antibacterial, diagnostics and imaging, and gene delivery. This review will focus on lipid nanosystems with a wide range of applications, taking into consideration their composition, properties, and physical parameters. However, designing suitable protocol for the physical evaluation of nanoparticles is still conflicting. The main obstacle is concerning the sensitivity, reproducibility, and reliability of the adopted methodology. Some important techniques are compared and discussed in this report. Particularly, a comparison between different techniques involved in (a) the morphologic characterization, such as Cryo-TEM, SEM, and X-ray; (b) the size measurement, such as dynamic light scattering, sedimentation field flow fractionation, and optical microscopy; and (c) surface properties, namely zeta potential measurement, is described. In addition, an amperometric tool in order to investigate antioxidant activity and the response of nanomaterials towards the skin membrane has been presented. Full article
(This article belongs to the Special Issue Advances in Characterization Methods for Drug Delivery Systems)
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