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Pharmaceutics
Volume 14
Issue 4
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Pharmaceutics, Volume 14, Issue 4 (April 2022) – 202 articles

Cover Story (view full-size image): Hyaluronic acid (HA), through its interactions with the cluster of differentiation 44 (CD44), acts as a potent modulator of the tumor microenvironment, creating extracellular stimuli for tumor growth, angiogenesis, invasion, and metastasis. An innovative antitumor strategy based on the development of a nanodevice for selective release of an inhibitor of the HA-CD44 interaction is presented. Computational analysis was performed to evaluate the interaction of the designed inhibitor with a CD44 binding site. Cell viability, efficiency, and selectivity of drug release under acidic conditions together with CD44 binding capacity, migration, and apoptotic effect were evaluated. Remarkably, the nanodevice generated a reduction in the dose required to achieve a significant therapeutic effect. View this paper.
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15 pages, 4066 KiB  
Article
A Novel CXCR4-Targeted Diphtheria Toxin Nanoparticle Inhibits Invasion and Metastatic Dissemination in a Head and Neck Squamous Cell Carcinoma Mouse Model
by Elisa Rioja-Blanco, Alberto Gallardo, Irene Arroyo-Solera, Patricia Álamo, Isolda Casanova, Ugutz Unzueta, Naroa Serna, Laura Sánchez-García, Miquel Quer, Antonio Villaverde, Esther Vázquez, Xavier León, Lorena Alba-Castellón and Ramon Mangues
Pharmaceutics 2022, 14(4), 887; https://doi.org/10.3390/pharmaceutics14040887 - 18 Apr 2022
Cited by 6 | Viewed by 2757
Abstract
Loco-regional recurrences and metastasis represent the leading causes of death in head and neck squamous cell carcinoma (HNSCC) patients, highlighting the need for novel therapies. Chemokine receptor 4 (CXCR4) has been related to loco-regional and distant recurrence and worse patient prognosis. In this [...] Read more.
Loco-regional recurrences and metastasis represent the leading causes of death in head and neck squamous cell carcinoma (HNSCC) patients, highlighting the need for novel therapies. Chemokine receptor 4 (CXCR4) has been related to loco-regional and distant recurrence and worse patient prognosis. In this regard, we developed a novel protein nanoparticle, T22-DITOX-H6, aiming to selectively deliver the diphtheria toxin cytotoxic domain to CXCR4+ HNSCC cells. The antimetastatic effect of T22-DITOX-H6 was evaluated in vivo in an orthotopic mouse model. IVIS imaging system was utilized to assess the metastatic dissemination in the mouse model. Immunohistochemistry and histopathological analyses were used to study the CXCR4 expression in the cancer cells, to evaluate the effect of the nanotoxin treatment, and its potential off-target toxicity. In this study, we report that CXCR4+ cancer cells were present in the invasive tumor front in an orthotopic mouse model. Upon repeated T22-DITOX-H6 administration, the number of CXCR4+ cancer cells was significantly reduced. Similarly, nanotoxin treatment effectively blocked regional and distant metastatic dissemination in the absence of systemic toxicity in the metastatic HNSCC mouse model. The repeated administration of T22-DITOX-H6 clearly abrogates tumor invasiveness and metastatic dissemination without inducing any off-target toxicity. Thus, T22-DITOX-H6 holds great promise for the treatment of CXCR4+ HNSCC patients presenting worse prognosis. Full article
(This article belongs to the Special Issue Novel Strategies for Cancer Targeted Delivery)
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Graphical abstract

Graphical abstract
Full article ">Figure 1
<p>CXCR4 expression in the tumor invasive front generated in the HNSCC orthotopic mouse model. (<b>A</b>) Representative CXCR4 and human-vimentin IHC images of the tumor budding showing the presence of CXCR4<sup>+</sup> cancer cells invading the surrounding tissue. Scale bars = 200 µm and 100 µm. (<b>B</b>) CXCR4 and human-vimentin immunofluorescence staining in the invasive front of the orthotopic tumor samples. Scale bars = 50 µm.</p> Full article ">Figure 2
<p>Anti-invasive T22-DITOX-H6 effect in the tumor front in a HNSCC orthotopic mouse model. (<b>A</b>) Representative human-vimentin IHC images of tumors collected from buffer- and T22-DITOX-H6-treated animals, showing a reduction in the number of human-vimentin-positive stained cells in the invasive front of the tumors after the nanotoxin treatment. Scale bar = 200 µm. (<b>B</b>) Quantification of the number of human-vimentin-positive stained cells in the tumor budding in the control and nanotoxin-treated tumors. (<b>C</b>) CXCR4 IHC analysis of the invasive front of tumors derived from control and nanotoxin-treated mice, displaying a reduction in the number of CXCR4<sup>+</sup> cells upon T22-DITOX-H6 treatment. Scale bar = 200 µm. (<b>D</b>) Quantification of the number of CXCR4-positive stained cells in the aforementioned CXCR4 IHC images. * <span class="html-italic">p</span> &lt; 0.05; <span class="html-italic">n</span> = 4 per group (total animal number 8). Statistical analysis was performed by Mann–Whitney test. Error bars indicate SEM.</p> Full article ">Figure 3
<p>T22-DITOX-H6 repeated administration reduces the occurrence of regional dissemination to the cervical lymph nodes in a HNSCC-disseminated mouse model. (<b>A</b>) Bioluminescence intensity (BLI) emitted by 74B-Luci cancer cells during the experiment in the buffer- and T22-DITOX-H6-treated animals. (<b>B</b>) Semi-quantification of the emitted BLI in the cervical lymph nodes (LNs) throughout the experiment in the control and treated groups. (<b>C</b>) Area under the curve (AUC) of the registered BLI emitted by cervical lymph nodes (LN) in the time course of the experiment for both control and nanotoxin-treated animals. (<b>D</b>) Percentage of the animals presenting cervical-lymph-node (LN) infiltration at the endpoint of the experiment in the buffer- and T22-DITOX-H6-treated groups. (<b>E</b>) Human vimentin IHC analysis of cervical lymph node samples from control and treated animals at the endpoint of the experiment (day 30 post-tumor-cell inoculation). Scale bars = 500 µm and 200 µm. (<b>F</b>) Representative images of the cervical lymph nodes (LN) from a buffer-treated animal (up) and a nanotoxin-treated animal (down) at euthanasia. Animals from the buffer-treated group presented macroscopic infiltrated lymph nodes. (<b>G</b>) Quantification of the area of the cervical lymph nodes observed in the human-vimentin IHC samples collected from buffer- and T22-DITOX-H6 groups. * <span class="html-italic">p</span> &lt; 0.05; ** <span class="html-italic">p</span> &lt; 0.01; *** <span class="html-italic">p</span> &lt; 0.001; <span class="html-italic">n</span> = 7 per group (total animal number 14). Statistical analysis was performed by Scheirer–Ray–Hare test, Mann–Whitney test, and Fisher’s test. Error bars indicate SEM.</p> Full article ">Figure 4
<p>T22-DITOX-H6 repeated administration inhibits distant metastatic dissemination to lungs and liver in a HNSCC-disseminated mouse model. (<b>A</b>) Representative human-vimentin IHC images of lung metastatic foci in samples obtained from buffer- and nanotoxin-treated mice. Scale bars = 100 µm. (<b>B</b>) Percentage of the animals from control and treated groups displaying lung metastases detected by human-vimentin IHC. (<b>C</b>) Quantification of the number of lung metastatic foci in each animal from the buffer- and T22-DITOX-H6-treated groups. (<b>D</b>) Human-vimentin IHC images showing the metastatic foci in the liver samples collected from control and treated mice. Scale bars = 100 µm. (<b>E</b>) Percentage of the animals from buffer- and T22-DITOX-H6 groups presenting liver metastases detected by human-vimentin IHC. (<b>F</b>) Quantification of the number of liver metastatic foci in each animal from the buffer- and nanotoxin-treated groups. ** <span class="html-italic">p</span> &lt; 0.01; *** <span class="html-italic">p</span> &lt; 0.001; <span class="html-italic">n</span> = 7 per group (total animal number 14). Statistical analysis was performed by Mann–Whitney test and Fisher’s test. Error bars indicate SEM.</p> Full article ">Figure 5
<p>Evaluation of the systemic toxicity derived from T22-DITOX-H6 administration in the HNSCC-disseminated mouse model. (<b>A</b>) Histopathological analysis by H&amp;E staining in liver, kidneys, and spleen samples collected from buffer- and T22-DITOX-H6-treated groups. Scale bars = 100 µm and 50 µm (zoom in) (<b>B</b>) Body weights of buffer- and nanotoxin-treated animals over the course of the experiment. Error bars indicate SEM.</p> Full article ">
20 pages, 801 KiB  
Review
An Overview of Cell Membrane Perforation and Resealing Mechanisms for Localized Drug Delivery
by Stephanie He, Davindra Singh and Brandon Helfield
Pharmaceutics 2022, 14(4), 886; https://doi.org/10.3390/pharmaceutics14040886 - 18 Apr 2022
Cited by 12 | Viewed by 3663
Abstract
Localized and reversible plasma membrane disruption is a promising technique employed for the targeted deposition of exogenous therapeutic compounds for the treatment of disease. Indeed, the plasma membrane represents a significant barrier to successful delivery, and various physical methods using light, sound, and [...] Read more.
Localized and reversible plasma membrane disruption is a promising technique employed for the targeted deposition of exogenous therapeutic compounds for the treatment of disease. Indeed, the plasma membrane represents a significant barrier to successful delivery, and various physical methods using light, sound, and electrical energy have been developed to generate cell membrane perforations to circumvent this issue. To restore homeostasis and preserve viability, localized cellular repair mechanisms are subsequently triggered to initiate a rapid restoration of plasma membrane integrity. Here, we summarize the known emergency membrane repair responses, detailing the salient membrane sealing proteins as well as the underlying cytoskeletal remodeling that follows the physical induction of a localized plasma membrane pore, and we present an overview of potential modulation strategies that may improve targeted drug delivery approaches. Full article
(This article belongs to the Special Issue Advances in Physics Methods for Drug Delivery)
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Figure 1
<p>Summary of known plasma membrane resealing mechanisms. (<b>a</b>) The tension–reduction hypothesis; (<b>b</b>) the patch hypothesis; (<b>c</b>) the ESCRT mechanism; and (<b>d</b>) exocytosis/endocytosis. See text for details.</p> Full article ">
10 pages, 2225 KiB  
Article
Anti-Psoriasis Effect of Diclofenac and Celecoxib Using the Tail Model for Psoriasis
by Diana Ana-Maria Nițescu, Horia Păunescu, Alina Elena Ștefan, Laurențiu Coman, Corneliu Cristian Georgescu, Andrei Constantin Stoian, Daniela Gologan, Ion Fulga and Oana Andreia Coman
Pharmaceutics 2022, 14(4), 885; https://doi.org/10.3390/pharmaceutics14040885 - 18 Apr 2022
Cited by 2 | Viewed by 2885
Abstract
Non-steroidal anti-inflammatory drugs (NSAIDs) showed effects in some hyperproliferative dermatologic pathologies. The aim of the study is the assessment of anti-psoriasis effect of diclofenac and celecoxib using a mice tail model. The topical application of substances on the proximal mice tails was performed [...] Read more.
Non-steroidal anti-inflammatory drugs (NSAIDs) showed effects in some hyperproliferative dermatologic pathologies. The aim of the study is the assessment of anti-psoriasis effect of diclofenac and celecoxib using a mice tail model. The topical application of substances on the proximal mice tails was performed for two weeks. The effects on the epidermal granular layer and mean epidermal thickness (excluding the stratum corneum) were evaluated using hematoxylin–eosin staining. Orthokeratosis degree and percentual drug activity were calculated. A positive control group treated with tretinoin and two negative controls (white soft paraffin and untreated mice) were used. Orthokeratosis degree significantly increased in all the NSAIDs groups (celecoxib 1%, 2% and diclofenac 1%, 2%) and in the tretinoin 0.05% group, versus negative controls. Celecoxib 1% and 2%, tretinoin 0.05% and white soft paraffin significantly increased mean epidermal thickness, versus untreated mice. The values obtained in the case of celecoxib 2% ointment regarding the orthokeratosis degree and percentual drug activity are providing premises for further investigations regarding this effect and the mechanisms of action involved. Celecoxib 2% had the greatest percentual drug activity and is a promising substance for the anti-psoriasis topical treatment. Along with the COX-2 inhibition, celecoxib might have an anti-psoriasis effect by other independent mechanisms. Full article
(This article belongs to the Special Issue Drugs in Dermatology: Topical Agent, Cosmetics, Dermatological Drug Delivery, Natural Product in Dermatology)
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Figure 1

Figure 1
<p>Negative control group section (untreated mice), hematoxylin–eosin staining, 10× objective.</p> Full article ">Figure 2
<p>Section with granular layer, hematoxylin–eosin staining, 40× objective.</p> Full article ">Figure 3
<p>Section group treated with 0.1 milliliter celecoxib 2% applied in the proximal part of the tail. The granular layer extends on the whole length scale, hematoxylin–eosin staining, 10× objective.</p> Full article ">Figure 4
<p>The orthokeratosis degree.</p> Full article ">Figure 5
<p>The percentual drug activity.</p> Full article ">Figure 6
<p>The mean epidermal thickness.</p> Full article ">
16 pages, 2968 KiB  
Article
β-Lactoglobulin-Modified Mesoporous Silica Nanoparticles: A Promising Carrier for the Targeted Delivery of Fenbendazole into Prostate Cancer Cells
by Maedeh Koohi Moftakhari Esfahani, Seyed Ebrahim Alavi, Peter J. Cabot, Nazrul Islam and Emad L. Izake
Pharmaceutics 2022, 14(4), 884; https://doi.org/10.3390/pharmaceutics14040884 - 18 Apr 2022
Cited by 17 | Viewed by 3413
Abstract
The clinical utilization of fenbendazole (FBZ) as a potential anticancer drug has been limited due to its low water solubility, which causes its low bioavailability. The development of a drug nanoformulation that includes the solubilizing agent as a drug carrier can improve solubility [...] Read more.
The clinical utilization of fenbendazole (FBZ) as a potential anticancer drug has been limited due to its low water solubility, which causes its low bioavailability. The development of a drug nanoformulation that includes the solubilizing agent as a drug carrier can improve solubility and bioavailability. In this study, Mobil Composition of Matter Number 48 (MCM-48) nanoparticles were synthesized and functionalized with succinylated β-lactoglobulin (BLG) to prevent early-burst drug release. The BLG-modified amine-functionalized MCM-48 (MCM-BLG) nanoparticles were loaded with FBZ to produce the drug nanoformulation (FBZ-MCM-BLG) and improved the water solubility and, consequently, its anticancer effects against human prostate cancer PC-3 cells. The prepared FBZ-MCM-BLG was characterized in terms of size, zeta potential, drug loading capacity, morphology, thermal and chemical analyses, drug release, cellular uptake, cell viability, cell proliferation, production of reactive oxygen species (ROS), and cell migration. The results demonstrated that the FBZ-MCM-BLG nanoparticles have a spherical morphology with a size and zeta potential of 369 ± 28 nm and 28 ± 0.4 mV, respectively. The drug loading efficiency of the new nanoformulation was 19%. The release of FBZ was pH-dependent; a maximum cumulative release of about 76 and 62% in 12 h and a burst release of 53 and 38% in the first 0.5 h was observed at pH 1.2 and 6.8, respectively. The prepared FBZ-MCM-BLG formulation demonstrated higher cytotoxicity effects against PC-3 cells by 5.6- and 1.8-fold, respectively, when compared to FBZ and FBZ-MCM nanoparticles. The new formulation also increased the production of ROS by 1.6- and 1.2-fold and inhibited the migration of PC-3 cells when compared to the FBZ and FBZ-MCM nanoparticles, respectively. Overall, FBZ-MCM-BLG nanoparticles improved FBZ delivery to PC-3 cells and have the potential to be evaluated for the treatment of prostate cancer following a comprehensive in vivo study. Full article
(This article belongs to the Special Issue Smart Drug Delivery Strategies Based on Porous Materials)
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Graphical abstract

Graphical abstract
Full article ">Figure 1
<p>(<b>A</b>) Size diagrams of MCM-48, MCM, FBZ-MCM, MCM-BLG, and FBZ-MCM-BLG nanoparticles. (<b>B</b>) Zeta potential of MCM-48, MCM, FBZ-MCM, MCM-BLG, and FBZ-MCM-BLG nanoparticles. (<b>C</b>) Size, polydispersity index (PDI), and zeta potential of the formulations. (<b>D</b>) Scanning electron microscopy (SEM) images and (<b>E</b>) transmission electron microscopy (TEM) images of MCM-48, MCM, FBZ-MCM, MCM-BLG, and FBZ-MCM-BLG nanoparticles. As the figure demonstrates, spherical, monodisperse, and homogenous nanoparticles were formed.</p> Full article ">Figure 2
<p>(<b>A</b>) TGA thermogram of FBZ, MCM-48, MCM, FBZ-MCM, MCM-BLG, and FBZ-MCM-BLG nanoparticles; (<b>B</b>) DSC thermogram of FBZ, MCM-48, MCM, FBZ-MCM, MCM-BLG, and FBZ-MCM-BLG nanoparticles; (<b>C</b>) N2 adsorption-desorption isotherms; and (<b>D</b>) pore size distributions of MCM and FBZ-MCM nanoparticles using the BET method.</p> Full article ">Figure 3
<p>XRD pattern of the synthesized MCM-48, MCM, FBZ-MCM, and FBZ-MCM-BLG nanoparticles.</p> Full article ">Figure 4
<p>Fourier transform infrared spectroscopy (FTIR) spectra of (<b>A</b>) MCM-48, MCM, and MCM-BLG; and (<b>B</b>) FBZ, FBZ-MCM, and FBZ-MCM-BLG nanoparticles. The peaks at 1622, 736, and 688 cm<sup>−1</sup> regions in the spectra of FBZ, FBZ-MCM, and FBZ-MCM-BLG nanoparticles prove that (i) FBZ was loaded into these nanoparticles and (ii) the chemical structure of the drug remained intact, meaning that FBZ was physically loaded into these NPs.</p> Full article ">Figure 5
<p>Drug release pattern from FBZ-MCM and FBZ-MCM-BLG nanoparticles, calculated at pH 1.2 and 6.8. One-way analysis of variance (ANOVA) and F tests were used for statistical analysis of the data. Additionally, the values of the slope (accumulative release/hour) for FBZ-MCM and FBZ-MCM-BLG at pH 1.2 were 5.0 and 4.2, respectively, whereas these values at pH 6.8 were 4.2 and 3.4, respectively. The data are expressed as mean ± SD (<span class="html-italic">n</span> = 3).</p> Full article ">Figure 6
<p>(<b>A</b>) Cellular uptake images of PC-3 cells after 4 h incubation with Cy5-MCM and Cy5-MCM-BLG nanoparticles. As the images show, higher fluorescence intensity was observed in the cells incubated with MCM-BLG nanoparticles. (<b>B</b>) Viability effects of FBZ-MCM and FBZ-MCM-BLG nanoparticles compared to FBZ on prostate cancer PC-3 cells after 48 h incubation. The data are expressed as mean ± SD (<span class="html-italic">n</span> = 3). (<b>C</b>) Cell proliferation effects of FBZ, FBZ-MCM, and FBZ-MCM-BLG nanoparticles on PC-3 cells over 5 days. (<b>D</b>) ROS generation in PC-3 cells after 6 h incubation with FBZ, FBZ-MCM, and FBZ-MCM-BLG nanoparticles. The results are expressed as mean ± SD from three independent experiments and were analyzed using a t-test: ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001.</p> Full article ">Figure 7
<p>(<b>A</b>,<b>B</b>) Effects of FBZ, FBZ-MCM, and FBZ-MCM-BLG nanoparticles on the migration and invasion of prostate cancer PC-3 cells before (0 h), 24 h after, and 48 h after cell treatment (×40 mag). Compared to the control group, FBZ, FBZ-MCM, and FBZ-MCM-BLG restrained cell migration, confirming the efficacy of FBZ, FBZ-MCM, and FBZ-MCM-BLG in inhibiting prostate cancer cell metastasis and invasion.</p> Full article ">
26 pages, 2464 KiB  
Review
Nanocarrier Drug Delivery Systems: Characterization, Limitations, Future Perspectives and Implementation of Artificial Intelligence
by Samar Zuhair Alshawwa, Abeer Ahmed Kassem, Ragwa Mohamed Farid, Shaimaa Khamis Mostafa and Gihan Salah Labib
Pharmaceutics 2022, 14(4), 883; https://doi.org/10.3390/pharmaceutics14040883 - 18 Apr 2022
Cited by 108 | Viewed by 8827
Abstract
There has been an increasing demand for the development of nanocarriers targeting multiple diseases with a broad range of properties. Due to their tiny size, giant surface area and feasible targetability, nanocarriers have optimized efficacy, decreased side effects and improved stability over conventional [...] Read more.
There has been an increasing demand for the development of nanocarriers targeting multiple diseases with a broad range of properties. Due to their tiny size, giant surface area and feasible targetability, nanocarriers have optimized efficacy, decreased side effects and improved stability over conventional drug dosage forms. There are diverse types of nanocarriers that have been synthesized for drug delivery, including dendrimers, liposomes, solid lipid nanoparticles, polymersomes, polymer–drug conjugates, polymeric nanoparticles, peptide nanoparticles, micelles, nanoemulsions, nanospheres, nanocapsules, nanoshells, carbon nanotubes and gold nanoparticles, etc. Several characterization techniques have been proposed and used over the past few decades to control and predict the behavior of nanocarriers both in vitro and in vivo. In this review, we describe some fundamental in vitro, ex vivo, in situ and in vivo characterization methods for most nanocarriers, emphasizing their advantages and limitations, as well as the safety, regulatory and manufacturing aspects that hinder the transfer of nanocarriers from the laboratory to the clinic. Moreover, integration of artificial intelligence with nanotechnology, as well as the advantages and problems of artificial intelligence in the development and optimization of nanocarriers, are also discussed, along with future perspectives. Full article
(This article belongs to the Special Issue Novel Nanoparticle-Based Treatment and Imaging Modalities)
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Figure 1
<p>Schematic representation of an asymmetric flow field flow fractionation channel equipped with a frit inlet (FI-AF4). Frit inlet flow propels sample components towards the accumulation wall, allowing their hydrodynamic relaxation without stopping their axial migration. Adapted with permission from [<a href="#B22-pharmaceutics-14-00883" class="html-bibr">22</a>], Elsevier, 2021.</p> Full article ">Figure 2
<p>Cryo-SEM of alginate (Sigma A0682) beads showing external bead surfaces for (<b>A</b>) a 2% alginate extrusion bead, (<b>B</b>) a 2% alginate emulsion bead and (<b>C</b>) a 5% alginate emulsion bead, as well as (<b>D</b>), a 5% alginate bead cut in half, with the cut surface facing the camera. The scale is the same for all images. Adapted with permission from [<a href="#B43-pharmaceutics-14-00883" class="html-bibr">43</a>], Wiley Online Library, 2012.</p> Full article ">Figure 3
<p>(<b>a</b>) TEM and (<b>b</b>) SEM images of lyophilized DLX-NLC. These micrographs revealed the nanoparticulate (80.17–127.73 nm) and spherical nature of DLX-NLC. Adapted with permission from [<a href="#B47-pharmaceutics-14-00883" class="html-bibr">47</a>], Elsevier, 2014.</p> Full article ">Figure 4
<p>Gamma scintigraphy images after intranasal administration (6 h) of (<b>a</b>) DLX-NLC suspension, (<b>b</b>) DLX solution. These images show the localization of DLX in different organs, including brain of rabbit. DLX-NLC exhibited better localization than DLX. Adapted with permission from [<a href="#B47-pharmaceutics-14-00883" class="html-bibr">47</a>], Elsevier, 2014.</p> Full article ">Figure 5
<p>Pharmaceutical nanotechnology challenges and current Limitations. FDA—Food and Drug Administration; EMA—European Medicines Agency; CDER—Center for Drug Evaluation and Research; GMP—Good Manufacturing Practices.</p> Full article ">
1 pages, 394 KiB  
Correction
Correction: Kazmi et al. Formulation and Evaluation of Kaempferol Loaded Nanoparticles against Experimentally Induced Hepatocellular Carcinoma: In Vitro and In Vivo Studies. Pharmaceutics 2021, 13, 2086
by Imran Kazmi, Fahad A. Al-Abbasi, Muhammad Afzal, Hisham N. Altayb, Muhammad Shahid Nadeem and Gaurav Gupta
Pharmaceutics 2022, 14(4), 882; https://doi.org/10.3390/pharmaceutics14040882 - 18 Apr 2022
Cited by 3 | Viewed by 1499
Abstract
In the original publication [...] Full article
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Figure 1

Figure 1
<p>FTIR spectra of the Kaempferol (pure drug) and the prepared Kaempferol nanoparticle.</p> Full article ">
21 pages, 5780 KiB  
Article
Oleyl Conjugated Histidine-Arginine Cell-Penetrating Peptides as Promising Agents for siRNA Delivery
by Muhammad Imran Sajid, Dindyal Mandal, Naglaa Salem El-Sayed, Sandeep Lohan, Jonathan Moreno and Rakesh Kumar Tiwari
Pharmaceutics 2022, 14(4), 881; https://doi.org/10.3390/pharmaceutics14040881 - 18 Apr 2022
Cited by 13 | Viewed by 3546
Abstract
Recent approvals of siRNA-based products motivated the scientific community to explore siRNA as a treatment option for several intractable ailments, especially cancer. The success of approved siRNA therapy requires a suitable and safer drug delivery agent. Herein, we report a series of oleyl [...] Read more.
Recent approvals of siRNA-based products motivated the scientific community to explore siRNA as a treatment option for several intractable ailments, especially cancer. The success of approved siRNA therapy requires a suitable and safer drug delivery agent. Herein, we report a series of oleyl conjugated histidine–arginine peptides as a promising nonviral siRNA delivery tool. The conjugated peptides were found to bind with the siRNA at N/P ratio ≥ 2 and demonstrated complete protection for the siRNA from early enzymatic degradation at N/P ratio ≥ 20. Oleyl-conjugated peptide -siRNA complexes were found to be noncytotoxic in breast cancer cells (MCF-7 and MDA-MB-231) and normal breast epithelial cells (MCF 10A) at N/P ratio of ~40. The oleyl-R3-(HR)4 and oleyl-R4-(HR)4 showed ~80-fold increased cellular uptake in MDA-MB-231 cells at N/P 40. Moreover, the conjugated peptides-siRNA complexes form nanocomplexes (~115 nm in size) and have an appropriate surface charge to interact with the cell membrane and cause cellular internalization. Furthermore, this study provides a proof-of-concept that oleyl-R5-(HR)4 can efficiently silence STAT-3 gene (~80% inhibition) in MDA-MB-231 cells with similar effectiveness to Lipofectamine. Further exploration of this approach holds a great promise in discovering a successful in vivo siRNA delivery agent with a favorable pharmacokinetic profile. Full article
(This article belongs to the Special Issue Peptide-Based Drug Delivery Systems)
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Graphical abstract

Graphical abstract
Full article ">Figure 1
<p>(<b>A</b>) Cytotoxicity of peptide/siRNA complex in MDA-MB-231 cells (<b>B</b>) Cytotoxicity of peptide/siRNA complex in MCF-7 cells (<b>C</b>) Cytotoxicity of peptide/siRNA complex in MCF-10A cells. Incubation time in each assay was 48 h. Graphs represent mean normalized values (based on No Treatment or NT group; <span class="html-italic">n</span> = 3). ns means nonsignificant. (if <span class="html-italic">p</span> &lt; 0.001 then ***, if <span class="html-italic">p</span> &lt; 0.0001 then ****). Error bars indicate standard deviation. Thirty-five percent <span class="html-italic">v</span>/<span class="html-italic">v</span> DMSO served as a positive control.</p> Full article ">Figure 1 Cont.
<p>(<b>A</b>) Cytotoxicity of peptide/siRNA complex in MDA-MB-231 cells (<b>B</b>) Cytotoxicity of peptide/siRNA complex in MCF-7 cells (<b>C</b>) Cytotoxicity of peptide/siRNA complex in MCF-10A cells. Incubation time in each assay was 48 h. Graphs represent mean normalized values (based on No Treatment or NT group; <span class="html-italic">n</span> = 3). ns means nonsignificant. (if <span class="html-italic">p</span> &lt; 0.001 then ***, if <span class="html-italic">p</span> &lt; 0.0001 then ****). Error bars indicate standard deviation. Thirty-five percent <span class="html-italic">v</span>/<span class="html-italic">v</span> DMSO served as a positive control.</p> Full article ">Figure 2
<p>Zeta potential values of peptide-siRNA complexes, where siRNA concentration in each complex in 50 nM and the concentration of the peptide varied according to their structure at N/P 40. Error bars indicate the standard deviation. Each experiment was performed independently in triplicate.</p> Full article ">Figure 3
<p>Cellular internalization of oleyl conjugated-siRNA complexes in MDA-MB-231. Bars indicate the mean fluorescent intensity of Alexa fluor-488 labeled siRNA. The error bars indicate ± SD (<span class="html-italic">n</span> = 3), ns means nonsignificant, * indicates <span class="html-italic">p</span>-value &lt; 0.05, *** indicate <span class="html-italic">p</span>-value &lt; 0.001.</p> Full article ">Figure 4
<p>Confocal microscopy images of MDA-MB-231 cells treated with oleyl-conjugated (HR)<sub>4</sub> peptides.</p> Full article ">Figure 5
<p>Representative images of gel retardation assay for evaluating binding affinity of the oleyl conjugated peptides with the siRNA.</p> Full article ">Figure 6
<p>Gel retardation assay of oleyl-conjugated peptides/siRNA complexes at N/P ratios ranging from 0 to 80. The bars indicate the mean intensity of the bands, representing percent siRNA bound to the peptides, quantified by Image Lab software. Error bars show the standard deviation (<span class="html-italic">n</span> = 3).</p> Full article ">Figure 7
<p>Representative images of gel retardation assay for evaluating the ability of the peptides to protect siRNA from 25% FBS after 24 h treatment. The gel was run after dissociating the peptides/siRNA complexes using a heparin competition assay.</p> Full article ">Figure 8
<p>Gel retardation assay of oleyl-conjugated peptides/siRNA complexes after 24 h exposure to 25% <span class="html-italic">v/v</span> FBS and dissociation of complexes using heparin competition assay at N/P ratios ranging from 0 to 80. The bars indicate the mean intensity of the bands, representing percent siRNA bound to the peptides, quantified by Image Lab software. Error bars show the standard deviation (<span class="html-italic">n</span> = 3).</p> Full article ">Figure 9
<p>Western blot image of STAT-3 protein silencing in MDA-MB-231 cells. Where GAPDH served as a house-keeping protein; [NT = Nontreated Cells, Lipo= Lipofectamine 2000, A = Oleyl-R<sub>2</sub>-(HR)<sub>4</sub>, B = Oleyl-R<sub>3</sub>-(HR)<sub>4</sub>, C = Oleyl-R<sub>4</sub>-(HR)<sub>4</sub>, D = Oleyl-R<sub>5</sub>-(HR)<sub>4</sub>].</p> Full article ">Figure 10
<p>Normalized STAT-3 expression quantified from the Western blot after 48 h of treatment with oleyl conjugated peptides-siRNA complexes at N/P 40. ns means nonsignificant, *** indicates a <span class="html-italic">p</span>-value &lt; 0.001, and **** indicate a <span class="html-italic">p</span>-value &lt; 0.0001.</p> Full article ">Scheme 1
<p>Synthesis of Oleyl-(HR)<sub>4</sub> peptide.</p> Full article ">
23 pages, 4903 KiB  
Article
Tolmetin Sodium Fast Dissolving Tablets for Rheumatoid Arthritis Treatment: Preparation and Optimization Using Box-Behnken Design and Response Surface Methodology
by Mahmoud M. A. Elsayed, Moustafa O. Aboelez, Bakheet E. M. Elsadek, Hatem A. Sarhan, Khaled Ali Khaled, Amany Belal, Ahmed Khames, Yasser A. Hassan, Amany A. Abdel-Rheem, Eslam B. Elkaeed, Mohamed Raafat and Mahmoud Elkot Mostafa Elsadek
Pharmaceutics 2022, 14(4), 880; https://doi.org/10.3390/pharmaceutics14040880 - 18 Apr 2022
Cited by 27 | Viewed by 4052
Abstract
Tolmetin sodium (TLM) is a non-steroidal anti-inflammatory drug (NSAIDs). TLM is used to treat inflammation, skeletal muscle injuries, and discomfort associated with bone disorders. Because of the delayed absorption from the gastro intestinal tract (GIT), the currently available TLM dosage forms have a [...] Read more.
Tolmetin sodium (TLM) is a non-steroidal anti-inflammatory drug (NSAIDs). TLM is used to treat inflammation, skeletal muscle injuries, and discomfort associated with bone disorders. Because of the delayed absorption from the gastro intestinal tract (GIT), the currently available TLM dosage forms have a rather protracted start to the effect, according to pharmacokinetic studies. The aim of this study was to create a combination for TLM fast dissolving tablets (TLM-FDT) that would boost the drug’s bioavailability by increasing pre-gastric absorption. The TLM-FDTs were developed using a Box-Behnken experimental design with varied doses of crospovidone (CP), croscarmellose sodium (CCS) as super-disintegrants, and camphor as a sublimating agent. In addition, the current study used response surface approach to explore the influence of various formulation and process factors on tablet qualities in order to verify an optimized TLM-FDTs formulation. The optimized TLM-FDTs formula was subsequently evaluated for its in vivo anti-inflammatory activity. TLM-FDTs have good friability, disintegration time, drug release, and wetting time, as well as fast disintegration and dissolution behavior. Significant increase in drug bioavailability and reliable anti-inflammatory efficacy were also observed, as evidenced by considerable reductions in paw thickness in rats following carrageenan-induced rat paw edema. For optimizing and analyzing the effect of super-disintegrants and sublimating agents in the TLM-FDTs formula, the three-factor, three-level full factorial design is a suitable tool. TLM-FDTs are a possible drug delivery system for enhancing TLM bioavailability and could be used to treat rheumatoid arthritis. Full article
(This article belongs to the Special Issue Development of Orally Dispersible Dosage Forms)
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<p>FTIR spectra of pure TLM (<b>A</b>); pure CP (<b>B</b>); pure CCS (<b>C</b>); pure camphor (<b>D</b>) and physical mixtures TLM/all excipients (<b>E</b>).</p> Full article ">Figure 2
<p>DSC thermograms of pure TLM (<b>A</b>); pure CP (<b>B</b>); pure CCS (<b>C</b>); pure camphor (<b>D</b>) and physical mixtures TLM/all excipients (<b>E</b>).</p> Full article ">Figure 3
<p>XRD profile of pure TLM (<b>A</b>); pure CP (<b>B</b>); pure CCS (<b>C</b>); pure camphor (<b>D</b>) and physical mixtures of TLM/all excipients (<b>E</b>).</p> Full article ">Figure 4
<p>The three-dimensional contour plot for the effect of total amounts of super-disintegrates (X<sub>1</sub> and X<sub>2</sub>) and percentage of sublimating agent (X<sub>3</sub>) on the percentage friability (Y<sub>1</sub>).</p> Full article ">Figure 5
<p>The three-dimensional contour plot for the effect of total amounts of super-disintegrates (X<sub>1</sub>), (X<sub>2</sub>) and percentage of sublimating agent (X<sub>3</sub>) on the disintegration time (Y<sub>2</sub>).</p> Full article ">Figure 6
<p>Shows 3D and response surface plots for the effect of super-disintegrates (X<sub>1</sub> and X<sub>2</sub>) and total amount of sublimating agents (X<sub>3</sub>) on the response Y<sub>3</sub> (% release).</p> Full article ">Figure 7
<p>The release profiles of TLM-FDT from different formulations. Release profiles of formulae 1–5 (<b>A</b>), release profiles of formulae 6–10 (<b>B</b>), and release profiles of formulae 11–15 (<b>C</b>).</p> Full article ">Figure 8
<p>Shows the 3D and response surface plot for the effect super-disintegrates (X<sub>1</sub> and X<sub>2</sub>) and total amount of sublimating agents (X<sub>3</sub>) on the response Y<sub>4</sub> (WT).</p> Full article ">Figure 9
<p>Changes in the paw volume at different time intervals in carrageenan-induced paw edema rat model. Data are presented as Mean ± SD (<span class="html-italic">n</span> = 6).</p> Full article ">Figure 10
<p>HPLC chromatograms of (<b>A</b>) TLM, (<b>B</b>) TLM in blank plasma, (<b>C</b>) plasma samples taken after injection of TLM-FDTs, and (<b>D</b>) blank plasma.</p> Full article ">Figure 11
<p>The mean concentration-time curve of TLM (μg/mL) in plasma after oral administration of TOLECTIN<sup>®</sup> tablets and TLM-FDTs in rats. Data are presented as the mean ± SD (<span class="html-italic">n</span> = 3).</p> Full article ">
17 pages, 2418 KiB  
Article
Transdermal Delivery of Metformin Using Dissolving Microneedles and Iontophoresis Patches for Browning Subcutaneous Adipose Tissue
by Mehrnaz Abbasi, Zhaoyang Fan, John A. Dawson and Shu Wang
Pharmaceutics 2022, 14(4), 879; https://doi.org/10.3390/pharmaceutics14040879 - 17 Apr 2022
Cited by 10 | Viewed by 4078
Abstract
Obesity is a serious public health problem that is strongly associated with increased multiple comorbidities such as diabetes, cardiovascular disease, and some types of cancer. While current anti-obesity treatments have various issues, locally transforming energy-storing white adipose tissue (WAT) into energy-burning brown-like/beige adipose [...] Read more.
Obesity is a serious public health problem that is strongly associated with increased multiple comorbidities such as diabetes, cardiovascular disease, and some types of cancer. While current anti-obesity treatments have various issues, locally transforming energy-storing white adipose tissue (WAT) into energy-burning brown-like/beige adipose tissue, the so-called browning of WAT, has been suggested to enhance obesity treatment efficiency with minimized side effects. Metformin is a first-line antidiabetes drug and a potent activator of AMP-activated protein kinase. Emerging evidence has suggested that metformin might enhance energy expenditure via the browning of WAT and hence reduce body weight. Subcutaneous WAT is easier to access and has a stronger browning potential than other WAT depots. In this study, we used dissolvable poly (lactic-co-glycolic acid) microneedles (MN) to deliver metformin to the subcutaneous WAT in obese C57BL/6J mice with the assistance of iontophoresis (INT), and then investigated metformin-induced WAT browning and its subsequent thermogenesis effects. Compared with MN alone or INT alone, MN + INT had better anti-obesity activity, as indicated by decreasing body weight and fat gain, increased energy expenditure, decreased fat pad size, and improved energy metabolism through the browning of WAT. Browning subcutaneous WAT by delivering metformin and other browning agents using this MN + INT approach might combat obesity in an effective, easy, and safe regimen. Full article
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Graphical abstract

Graphical abstract
Full article ">Figure 1
<p>Microneedle fabrication and characteristics. (<b>A</b>) Schematic illustration of the process of fabricating dissolving PLGA MN patches. (<b>B</b>) MPatch microneedle mold. (<b>C</b>) Fabricated MN patch overview. (<b>D</b>) DID-loaded MN and (<b>E</b>) Rhod-PE-loaded MN. SEM micrographs of MN patches. (<b>F</b>) Length and base of the mold. (<b>G</b>) Length and base of the needles in 800 μm MN. (<b>H</b>) Intact 800 μm MN. (<b>I</b>) Intact 800 μm MN.</p> Full article ">Figure 2
<p>In vivo fluorescence imaging and biodistribution of DID dye using MN patches and INT. IVIS images of (<b>A</b>) the biodistribution of DID dye using different treatments: Control, INT (DID) alone, MN (DID) alone, and MN (DID)+ INT in C57BL/6J mice and dissected organs. (<b>B</b>) Quantified fluorescence intensity of the delivered DID dye. Images are representatives of three independent experiments. Data were calculated from three independent experiments and expressed as means ± SEM.</p> Full article ">Figure 3
<p>Anti-obesity and metabolic effects of metformin. (<b>A</b>) Food intake, body weight, fat %, and lean %; (<b>B</b>) blood glucose levels and glucose tolerance test area under the curve; (<b>C</b>) tissue weight of GWAT and IgWAT; (<b>D</b>) metformin content in the liver and IgWAT; (<b>E</b>) adipocyte size; (<b>F</b>) metabolic activity parameters. (*) Lower blood glucose. Values are means ± SEM, <span class="html-italic">n</span> = 5 per treatment. Bars without a common superscript differ at <span class="html-italic">p</span> &lt; 0.05.</p> Full article ">Figure 4
<p>Browning effects, and thermogenic, lipogenic, inflammatory and glucogenic gene expression. Reciprocal intensity of the chromogen staining and representative immunohistochemistry staining images of IgWAT (scale bar: 200 µm): (<b>A</b>) UCP1, (<b>B</b>) AMPK, and (<b>C</b>) pAMPK. IgWAT mRNA level of (<b>D</b>) thermogenic markers: UCP1, ELOVL3, PRDM16, TMEM26, CIDEA, ZIC1, PGC1α, and CD137; (<b>E</b>) lipogenic (ACC1 and leptin) and inflammatory (MCP1 and TNFα) markers. Liver mRNA levels of (<b>F</b>) inflammatory (MCP1 and TNFα) and glucogenic (PEPCK and GLUT2) markers. Values are means ± SEM, <span class="html-italic">n</span> = 5 per treatment. Bars without a common superscript differ at <span class="html-italic">p</span> &lt; 0.05.</p> Full article ">
31 pages, 922 KiB  
Systematic Review
Systematic Review of Pharmacogenetics of ABC and SLC Transporter Genes in Acute Myeloid Leukemia
by Juan Eduardo Megías-Vericat, David Martínez-Cuadrón, Antonio Solana-Altabella, José Luis Poveda and Pau Montesinos
Pharmaceutics 2022, 14(4), 878; https://doi.org/10.3390/pharmaceutics14040878 - 17 Apr 2022
Cited by 9 | Viewed by 3260
Abstract
Antineoplastic uptake by blast cells in acute myeloid leukemia (AML) could be influenced by influx and efflux transporters, especially solute carriers (SLCs) and ATP-binding cassette family (ABC) pumps. Genetic variability in SLC and ABC could produce interindividual differences in clinical outcomes. A systematic [...] Read more.
Antineoplastic uptake by blast cells in acute myeloid leukemia (AML) could be influenced by influx and efflux transporters, especially solute carriers (SLCs) and ATP-binding cassette family (ABC) pumps. Genetic variability in SLC and ABC could produce interindividual differences in clinical outcomes. A systematic review was performed to evaluate the influence of SLC and ABC polymorphisms and their combinations on efficacy and safety in AML cohorts. Anthracycline intake was especially influenced by SLCO1B1 polymorphisms, associated with lower hepatic uptake, showing higher survival rates and toxicity in AML studies. The variant alleles of ABCB1 were related to anthracycline intracellular accumulation, increasing complete remission, survival and toxicity. Similar findings have been suggested with ABCC1 and ABCG2 polymorphisms. Polymorphisms of SLC29A1, responsible for cytarabine uptake, demonstrated significant associations with survival and response in Asian populations. Promising results were observed with SLC and ABC combinations regarding anthracycline toxicities. Knowledge of the role of transporter pharmacogenetics could explain the differences observed in drug disposition in the blast. Further studies including novel targeted therapies should be performed to determine the influence of genetic variability to individualize chemotherapy schemes. Full article
(This article belongs to the Special Issue The Role of SLC and ABC Transporters in Anti-cancer Drug Delivery)
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<p>Summary of evidence search and selection.</p> Full article ">Figure 2
<p>Key candidate genes involved in drug transport in acute myeloid leukemia.</p> Full article ">
21 pages, 4547 KiB  
Article
Enhanced In Vitro Antiviral Activity of Hydroxychloroquine Ionic Liquids against SARS-CoV-2
by Francisco Faísca, Vanessa Correia, Željko Petrovski, Luís C. Branco, Helena Rebelo-de-Andrade and Miguel M. Santos
Pharmaceutics 2022, 14(4), 877; https://doi.org/10.3390/pharmaceutics14040877 - 17 Apr 2022
Cited by 6 | Viewed by 3133
Abstract
The development of effective antiviral drugs against SARS-CoV-2 is urgently needed and a global health priority. In light of the initial data regarding the repurposing of hydroxychloroquine (HCQ) to tackle this coronavirus, herein we present a quantitative synthesis and spectroscopic and thermal characterization [...] Read more.
The development of effective antiviral drugs against SARS-CoV-2 is urgently needed and a global health priority. In light of the initial data regarding the repurposing of hydroxychloroquine (HCQ) to tackle this coronavirus, herein we present a quantitative synthesis and spectroscopic and thermal characterization of seven HCQ room temperature ionic liquids (HCQ-ILs) obtained by direct protonation of the base with two equivalents of organic sulfonic, sulfuric and carboxylic acids of different polarities. Two non-toxic and hydrophilic HCQ-ILs, in particular, [HCQH2][C1SO3]2 and [HCQH2][GlcCOO]2, decreased the virus-induced cytopathic effect by two-fold in comparison with the original drug, [HCQH2][SO4]. Despite there being no significant differences in viral RNA production between the three compounds, progeny virus production was significantly affected (p < 0.05) by [HCQH2][GlcCOO]2. Overall, the data suggest that the in vitro antiviral activities of the HCQ-ILs are most likely the result of specific intra- and intermolecular interactions and not so much related with their hydrophilic or lipophilic character. This work paves the way for the development of future novel ionic formulations of hydroxychloroquine with enhanced physicochemical properties. Full article
(This article belongs to the Special Issue Therapeutic Formulations of Repurposed Drugs against COVID-19)
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<p>FTIR-ATR spectra of [HCQH<sub>2</sub>][C<sub>1</sub>SO<sub>3</sub>]<sub>2</sub> (<b>3</b>), HCQ (<b>2</b>), C<sub>1</sub>SO<sub>3</sub>K and C<sub>1</sub>SO<sub>3</sub>H between ν 2000–400 cm<sup>−1</sup>.</p> Full article ">Figure 2
<p>Comparative cytotoxicity of the (<b>A</b>) different compounds (HCQ-ILs and parental drug) and (<b>B</b>) counterions in non-infected Vero E6 cells, evaluated by the amount of ATP present in cultures (CellTiter-Glo<sup>®</sup> Luminescent Cell Viability Assay) and expressed in relative values compared to vehicle control (DMEM maintenance medium alone or with 0.8% DMSO). Data are plotted as mean percent values across three independent experiments with triplicate measurements. The error bars represent the standard deviation of the mean (note: some error bars are shorter than the height of the symbol). Graphical representations were generated using GraphPad Prism software version 9.2.0 for Mac (GraphPad Software, San Diego, CA, USA).</p> Full article ">Figure 3
<p>Inhibition of SARS-CoV-2 replication based on virus-induced cytopathic effect (CPE) at 72 h post-infection (hpi) in Vero E6 cells treated with serial non-toxic concentrations of compounds (<b>A</b>) or anion salts (<b>B</b>). CPE was measured by calculating the number of live cells via a CellTiter-Glo<sup>®</sup> Luminescent Cell Viability Assay. Results are expressed in relation to virus control (untreated infected cells). Data are plotted as mean percent values across three and two independent experiments with triplicate measurements, respectively. Data were plotted and generated as indicated in <a href="#pharmaceutics-14-00877-f002" class="html-fig">Figure 2</a>.</p> Full article ">Figure 4
<p>Dose–response curves for the two HCQ-OSILs identified as promising and for the parental drug (<b>1</b>) based on viral RNA (vRNA) transcription (<b>A</b>) and progeny production (<b>B</b>). RNA transcription was assessed by quantification of virus yield using qRT-PCR assay (RdRp gene) and progeny production was determined by calculation of the infectious virus titer by TCID<sub>50</sub> assay (TCID<sub>50</sub>/mL), both at 48 hpi in cell supernatants. Results are expressed in relative values compared to virus control. Data were plotted and generated as indicated in <a href="#pharmaceutics-14-00877-f002" class="html-fig">Figure 2</a>. Four independent experiments were carried out on vRNA transcription assays (<b>A</b>).</p> Full article ">Scheme 1
<p>Methodology for the synthesis of the HCQ-ILs.</p> Full article ">
14 pages, 2145 KiB  
Article
Influence of Formulation Factors, Process Parameters, and Selected Quality Attributes on Carvedilol Release from Roller-Compacted Hypromellose-Based Matrix Tablets
by Aleša Dular Vovko, Bor Hodžić, Tina Brec, Grega Hudovornik and Franc Vrečer
Pharmaceutics 2022, 14(4), 876; https://doi.org/10.3390/pharmaceutics14040876 - 16 Apr 2022
Cited by 6 | Viewed by 2457
Abstract
The importance of roller compaction is recently increasing. This study evaluates the combined effects of formulation factors, process parameters, and selected quality attributes on drug release from roller-compacted hypromellose-based matrix tablets containing carvedilol as a model drug. The influence of selected factors was [...] Read more.
The importance of roller compaction is recently increasing. This study evaluates the combined effects of formulation factors, process parameters, and selected quality attributes on drug release from roller-compacted hypromellose-based matrix tablets containing carvedilol as a model drug. The influence of selected factors was statistically assessed and good predictive models were developed for various time points of the release profile. The results show that the release profile is mostly affected by the particle size distribution of granules and roll speed. This indicates that the roller compaction process has a major impact on drug release, which is also formulation dependent. A higher d50 and lower d90 value of spatial filtering technique-based particle size distribution results, a lower roll speed, increased hypromellose content, using microcrystalline cellulose as a filler, and higher tablet hardness, resulted in a decrease in the drug release rate. On the other hand, the effect of the roll pressure, size of screen apertures, and d10 values on drug release was insignificant. The significance of the factors was further explained by granule shape, their porosity, and friability evaluation, and by compressibility and compactibility studies of compression mixtures. Additionally, the spatial filtering technique demonstrated to be a promising tool in controlling the roller compaction process. Full article
(This article belongs to the Special Issue Feature Papers in Physical Pharmacy and Formulation)
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<p>Time dependence of coded coefficients of predictive models for significant factors (hypromellose content, filler type, roll speed, d50, d90, and tablet hardness).</p> Full article ">Figure 2
<p>Effect of particle size distribution and tablet hardness on % drug release from matrix tablets containing lactose as a filler.</p> Full article ">Figure 3
<p>Compressibility plot of selected compression mixtures containing lactose as a filler.</p> Full article ">Figure 4
<p>Compactibility plot of selected compression mixtures containing lactose as a filler.</p> Full article ">Figure 5
<p>Compressibility plot of selected compression mixtures containing microcrystalline cellulose (MCC) as a filler.</p> Full article ">Figure 6
<p>Compactibility plot of selected compression mixtures containing microcrystalline cellulose (MCC) as a filler.</p> Full article ">Figure 7
<p>Effect of roll speed and tablet hardness on % drug release from matrix tablets containing MCC as a filler.</p> Full article ">
25 pages, 4315 KiB  
Article
Tailoring of Selenium-Plated Novasomes for Fine-Tuning Pharmacokinetic and Tumor Uptake of Quercetin: In Vitro Optimization and In Vivo Radiobiodistribution Assessment in Ehrlich Tumor-Bearing Mice
by Heba M. Aboud, Amal K. Hussein, Abdallah Z. Zayan, Tarek Saad Makram, Mona O. Sarhan and Dina M. El-Sharawy
Pharmaceutics 2022, 14(4), 875; https://doi.org/10.3390/pharmaceutics14040875 - 16 Apr 2022
Cited by 14 | Viewed by 2703
Abstract
Quercetin (QRC) is a bioflavonoid with anti-inflammatory, antioxidant, and anticancer activities, yet QRC poor bioavailability has hampered its clinical implementation. The aim of the current work was to harness novasomes (NOVs), free fatty acid enriched vesicles, as a novel nano-cargo for felicitous QRC [...] Read more.
Quercetin (QRC) is a bioflavonoid with anti-inflammatory, antioxidant, and anticancer activities, yet QRC poor bioavailability has hampered its clinical implementation. The aim of the current work was to harness novasomes (NOVs), free fatty acid enriched vesicles, as a novel nano-cargo for felicitous QRC delivery with subsequent functionalization with selenium (SeNOVs), to extend the systemic bio-fate of NOVs and potentiate QRC anticancer efficacy through the synergy with selenium. QRC-NOVs were primed embedding oleic acid, Brij 35, and cholesterol adopting thin-film hydration technique according to Box–Behnken design. Employing Design-Expert® software, the impact of formulation variables on NOVs physicochemical characteristics besides the optimum formulation election were explored. Based on the optimal NOVs formulation, QRC-SeNOVs were assembled via electrostatic complexation/in situ reduction method. The MTT cytotoxicity assay of the uncoated, and coated nanovectors versus crude QRC was investigated in human rhabdomyosarcoma (RD) cells. The in vivo pharmacokinetic and biodistribution studies after intravenous administrations of technetium-99m (99mTc)-labeled QRC-NOVs, QRC-SeNOVs, and QRC-solution were scrutinized in Ehrlich tumor-bearing mice. QRC-NOVs and QRC-SeNOVs disclosed entrapment efficiency of 67.21 and 70.85%, vesicle size of 107.29 and 129.16 nm, ζ potential of −34.71 and −43.25 mV, and accumulatively released 43.26 and 31.30% QRC within 24 h, respectively. Additionally, QRC-SeNOVs manifested a far lower IC50 of 5.56 μg/mL on RD cells than that of QRC-NOVs (17.63 μg/mL) and crude QRC (38.71 μg/mL). Moreover, the biodistribution study elicited higher preferential uptake of 99mTc-QRC-SeNOVs within the tumorous tissues by 1.73- and 5.67-fold as compared to 99mTc-QRC-NOVs and 99mTc-QRC-solution, respectively. Furthermore, the relative uptake efficiency of 99mTc-QRC-SeNOVs was 5.78, the concentration efficiency was 4.74 and the drug-targeting efficiency was 3.21. Hence, the engineered QRC-SeNOVs could confer an auspicious hybrid nanoparadigm for QRC delivery with fine-tuned pharmacokinetics, and synergized antitumor traits. Full article
(This article belongs to the Section Nanomedicine and Nanotechnology)
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<p>Response surface 3D plot for the effect of oleic acid (<span class="html-italic">X</span><sub>1</sub>), Brij 35 (<span class="html-italic">X</span><sub>2</sub>), and cholesterol (<span class="html-italic">X</span><sub>3</sub>) concentrations on (<b>a</b>) EE% (<span class="html-italic">Y</span><sub>1</sub>), (<b>b</b>) vesicle size (<span class="html-italic">Y</span><sub>2</sub>), and (<b>c</b>) Q<sub>24h</sub> (<span class="html-italic">Y</span><sub>3</sub>).</p> Full article ">Figure 2
<p>(<b>a</b>) UV–Vis spectra of QRC-NOVs, QRC-SeNOVs, and selenium nanoparticles; (<b>b</b>) FT-IR spectra of QRC-NOVs, and QRC-SeNOVs.</p> Full article ">Figure 3
<p>Transmission electron photomicrographs of (<b>a</b>) QRC-NOVs, and (<b>b</b>) QRC-SeNOVs.</p> Full article ">Figure 4
<p>Effect of storage on EE%, vesicle size, and ζ potential of (<b>a</b>) QRC-NOVs, and (<b>b</b>) QRC-SeNOVs.</p> Full article ">Figure 5
<p>In vitro cytotoxicity of crude QRC, QRC-NOVs, and QRC-SeNOVs against RD cells via MTT assay after 48 h incubation. Data expressed as mean ± SD (<span class="html-italic">n</span> = 3). <sup>a</sup> <span class="html-italic">p</span> &lt; 0.05 versus free QRC. <sup>b</sup> <span class="html-italic">p</span> &lt; 0.05 versus QRC-NOVs.</p> Full article ">Figure 6
<p>Effect of different conditions on the RLY of the <sup>99m</sup>Tc-QRC complex: (<b>a</b>) sodium dithionite amount, (<b>b</b>) QRC amount, and (<b>c</b>) reaction time.</p> Full article ">Figure 7
<p>QRC blood concentration at various time intervals following i.v. administrations of <sup>99m</sup>Tc-QRC solution, <sup>99m</sup>Tc-QRC-NOVs, and <sup>99m</sup>Tc-QRC-SeNOVs in Ehrlich tumor-bearing adult male Swiss albino mice, mean ± SD, <span class="html-italic">n</span> = 3.</p> Full article ">Figure 8
<p>QRC tumor accumulation at various time intervals following i.v. administrations of <sup>99m</sup>Tc-QRC solution, <sup>99m</sup>Tc-QRC-NOVs, and <sup>99m</sup>Tc-QRC-SeNOVs in Ehrlich tumor-bearing adult male Swiss albino mice, mean ± SD, <span class="html-italic">n</span> = 3.</p> Full article ">
15 pages, 716 KiB  
Review
Reptiles as Promising Sources of Medicinal Natural Products for Cancer Therapeutic Drugs
by Soon Yong Park, Hyeongrok Choi and Jin Woong Chung
Pharmaceutics 2022, 14(4), 874; https://doi.org/10.3390/pharmaceutics14040874 - 16 Apr 2022
Cited by 5 | Viewed by 2635
Abstract
Natural products have historically played an important role as a source of therapeutic drugs for various diseases, and the development of medicinal natural products is still a field with high potential. Although diverse drugs have been developed for incurable diseases for several decades, [...] Read more.
Natural products have historically played an important role as a source of therapeutic drugs for various diseases, and the development of medicinal natural products is still a field with high potential. Although diverse drugs have been developed for incurable diseases for several decades, discovering safe and efficient anticancer drugs remains a formidable challenge. Reptiles, as one source of Asian traditional medicines, are known to possess anticancer properties and have been used for a long time without a clarified scientific background. Recently, it has been reported that extracts, crude peptides, sera, and venom isolated from reptiles could effectively inhibit the survival and proliferation of various cancer cells. In this article, we summarize recent studies applying ingredients derived from reptiles in cancer therapy and discuss the difficulties and prospective development of natural product research. Full article
(This article belongs to the Special Issue Natural Product-Inspired Approaches for Cancer Diagnosis and Therapy)
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<p>Schematic image of anticancer effect mechanisms of reptile-derived components.</p> Full article ">
16 pages, 2384 KiB  
Article
Polymeric Nanocapsules Containing Fennel Essential Oil: Their Preparation, Physicochemical Characterization, Stability over Time and in Simulated Gastrointestinal Conditions
by Giuseppe Granata, Carla Riccobene, Edoardo Napoli and Corrada Geraci
Pharmaceutics 2022, 14(4), 873; https://doi.org/10.3390/pharmaceutics14040873 - 16 Apr 2022
Cited by 14 | Viewed by 3490
Abstract
Plant essential oils, a source of biologically active compounds, represent a promising segment in the pharmaceutical market. However, their volatility, hydrophobicity, poor stability, and low toxicity limit direct use in pharmaceutical-related applications. Nanoencapsulation is a technique that allows overcoming these obstacles by improving [...] Read more.
Plant essential oils, a source of biologically active compounds, represent a promising segment in the pharmaceutical market. However, their volatility, hydrophobicity, poor stability, and low toxicity limit direct use in pharmaceutical-related applications. Nanoencapsulation is a technique that allows overcoming these obstacles by improving bioaccessibility and bioavailability. Nanocapsules (NCs) based on biodegradable and biocompatible poly(ɛ-caprolactone) containing Foeniculum vulgare Mill. essential oil (FEO), known for its biological activities, were successfully prepared by interfacial deposition of the preformed polymer method. The composition of FEO (trans-anethole chemotype) was determined by gas chromatography analyses. The FEO presence inside the NCs was confirmed by nuclear magnetic resonance experiments. The FEO-NCs showed nanometer size (210 nm), low polydispersity index (0.10), negative zeta potential (−15 mV), non-Newtonian rheological behavior, and high efficiency of encapsulation (93%). Moreover, parameters such as FEO-NC particle size, bioactive compound retention, and FEO composition were monitored for 30 days at storage temperatures of 4 and 40 °C, confirming the robustness of the nanosystem. Finally, FEO-NCs were resistant to the simulated gastric digestion and showed an effective bioaccessibility of 29% in simulated intestinal digestion. Based on the results obtained, this FEO-NC nanosystem could find interesting applications in the nutraceutical and pharmaceutical sectors. Full article
(This article belongs to the Special Issue Essential Oils in Pharmaceutical Products)
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Graphical abstract

Graphical abstract
Full article ">Figure 1
<p>Z-average diameter (<b>a</b>) and PDI (<b>b</b>) of FEO-NCs over time (storage at 4 and 40 °C).</p> Full article ">Figure 2
<p>Intensity weighted particle hydrodynamic diameter (<span class="html-italic">D<sub>H</sub></span>, nm) distribution of FEO-NC suspension: freshly prepared, stored at 4 and 40 °C for 30 days.</p> Full article ">Figure 3
<p>Zeta potential (<b>a</b>) and FEO retention (<b>b</b>) of FEO-NCs over time (storage at 4 and 40 °C).</p> Full article ">Figure 4
<p><sup>1</sup>H-NMR of FEO-NC nanosuspension in 450 µL of D<sub>2</sub>O: black line is the freshly prepared sample; blue line is the same sample after 30 days; green line is the sample with the addition of 200 µL of acetone-<span class="html-italic">d</span><sub>6</sub>; red line is the sample with the addition of 450 µL of acetone-<span class="html-italic">d</span><sub>6</sub>.</p> Full article ">Figure 5
<p>Apparent viscosity variation of the FEO-NC suspension as a function of shear rate. The red dashed line was obtained by fitting the viscosity variation using power law model.</p> Full article ">Figure 6
<p><span class="html-italic">Trans</span>-anethole variation by GC-FID (relative percentage) over time and storage at 4 and 40 °C.</p> Full article ">Figure 7
<p>(<b>Left</b>) Intensity weighted particle <span class="html-italic">D<sub>H</sub></span> (nm) distribution of FEO-NCs: not treated sample, after simulated gastric digestion (SGD), before and after neutralization. (<b>Right</b>) Physicochemical features after SGD (<b>a</b>); <span class="html-italic">trans</span>-anethole retention percentage after SGD (<b>b</b>).</p> Full article ">
39 pages, 11656 KiB  
Review
Recent Fabrication Methods to Produce Polymer-Based Drug Delivery Matrices (Experimental and In Silico Approaches)
by Anna Procopio, Elena Lagreca, Rezvan Jamaledin, Sara La Manna, Brunella Corrado, Concetta Di Natale and Valentina Onesto
Pharmaceutics 2022, 14(4), 872; https://doi.org/10.3390/pharmaceutics14040872 - 15 Apr 2022
Cited by 22 | Viewed by 4027
Abstract
The study of novel drug delivery systems represents one of the frontiers of the biomedical research area. Multi-disciplinary scientific approaches combining traditional or engineered technologies are used to provide major advances in improving drug bioavailability, rate of release, cell/tissue specificity and therapeutic index. [...] Read more.
The study of novel drug delivery systems represents one of the frontiers of the biomedical research area. Multi-disciplinary scientific approaches combining traditional or engineered technologies are used to provide major advances in improving drug bioavailability, rate of release, cell/tissue specificity and therapeutic index. Biodegradable and bio-absorbable polymers are usually the building blocks of these systems, and their copolymers are employed to create delivery components. For example, poly (lactic acid) or poly (glycolic acid) are often used as bricks for the production drug-based delivery systems as polymeric microparticles (MPs) or micron-scale needles. To avoid time-consuming empirical approaches for the optimization of these formulations, in silico-supported models have been developed. These methods can predict and tune the release of different drugs starting from designed combinations. Starting from these considerations, this review has the aim of investigating recent approaches to the production of polymeric carriers and the combination of in silico and experimental methods as promising platforms in the biomedical field. Full article
(This article belongs to the Special Issue Polymer-Based Matrices for Drug Delivery: In Vitro, In Vivo and In Silico Approaches)
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<p>Schematic representation of the structure of nanocapsules and nanospheres. Created with <a href="http://BioRender.com" target="_blank">BioRender.com</a> (accessed on 23 December 2021).</p> Full article ">Figure 2
<p>Schematic illustration of the most common designs used to make droplets with microfluidic devices. Flow-focusing (<b>a</b>): the two phases meet at the junction where the continuous phase pinches off droplets of the dispersed phase from the bottom and the top. T-junction (<b>b</b>), where both phases meet at a T-shaped junction and the shear stress created by the continuous phase causes droplet formation. (<b>c</b>) Droplet formation using a coaxial geometry, where the continuous phases completely surround the dispersed phase in three dimensions to create droplets. Created with <a href="http://BioRender.com" target="_blank">BioRender.com</a> (accessed on 24 December 2021).</p> Full article ">Figure 3
<p>(<b>a</b>) Example of a flow-focusing chip to produce photocurable droplets containing the monomers (TPM and DDMA) and the photoinitiator in the continuous phase (PVA solution). Under UV-irradiation, photopolymerization is initiated. Reprinted from reference [<a href="#B97-pharmaceutics-14-00872" class="html-bibr">97</a>]. Copyright 2018, Scientific Reports. (<b>b</b>) The fabrication process of the T-junction chip to generate droplets by varying oil viscosity. (<b>c</b>) A photograph of the experimental setup. Reprinted from reference [<a href="#B100-pharmaceutics-14-00872" class="html-bibr">100</a>], Copyright 2019, Micromachines.</p> Full article ">Figure 4
<p>(<b>a</b>) Schematic illustration of insertion-responsive microneedles. (<b>b</b>) Describing the dimensions of different structures. (<b>c</b>) Schematic illustration of the transverse compression test. Reprinted from [<a href="#B108-pharmaceutics-14-00872" class="html-bibr">108</a>] 2018, Royal society of chemistry.</p> Full article ">Figure 5
<p>(<b>a</b>) Schematic illustrations of compositions and properties of ice microneedles. (<b>b</b>) Schemes of the fabrication process of ice microneedles. (<b>c</b>) Image of an ice microneedle patch. (<b>d</b>) Digital images of agarose after penetration of unfrozen water microneedles (dyed green), GelMA microneedles (dyed yellow), Alg microneedles (dyed blue), and Matrigel microneedles (dyed red); digital images of agarose after penetration of ice microneedles; digital images of porcine skins after penetration of ice microneedles. Reprinted from [<a href="#B112-pharmaceutics-14-00872" class="html-bibr">112</a>], 2020, Wiley.</p> Full article ">Figure 6
<p>(<b>a</b>) Schematic representation of a classical electrospinning system. Reprinted from Fadil et al. [<a href="#B119-pharmaceutics-14-00872" class="html-bibr">119</a>], with permission under Polymers license. (<b>b</b>) Stroboscopic image of a polymeric jet, generated during electrospinning. Reprinted from [<a href="#B120-pharmaceutics-14-00872" class="html-bibr">120</a>] with permission under Polymer. (<b>c</b>) SEM image of the generated polymeric nanofibers. Reprinted from [<a href="#B120-pharmaceutics-14-00872" class="html-bibr">120</a>] with permission under Polymer. (<b>d</b>) Representation of the resulting nanofiber structures from different polymer feed solutions loaded for the electrospinning. Reprinted from Nikmaram et al. [<a href="#B121-pharmaceutics-14-00872" class="html-bibr">121</a>] with permission under RSC license. (<b>e</b>) Schematic representation of the impact of gravity on the resulting nanofibers in different orientations of the electrospinning system. Adapted from Nagam et al. [<a href="#B122-pharmaceutics-14-00872" class="html-bibr">122</a>] with permission under Fibers license.</p> Full article ">Figure 7
<p>(<b>a</b>) Schematic representation of the system employed for fabrication of the magnetic fibers loaded with iron oxide nanocubes (IONCs) and DOXO, and confocal images of nanofibers with (15–23 nm) and without nanoparticles. Reprinted from [<a href="#B137-pharmaceutics-14-00872" class="html-bibr">137</a>] with permission under Journal of colloid and interface science license. (<b>b</b>) Schematic representation of the new proposed spinneret, with its digital photograph and SEM images of the obtained nanofibers from (<b>a1</b>) the traditional process (N1), and (<b>b1</b>) new ones (N2). Reprinted from [<a href="#B138-pharmaceutics-14-00872" class="html-bibr">138</a>].</p> Full article ">Figure 8
<p>Main geometric parameters for (<b>a</b>) nanocapsule (reprinted from [<a href="#B150-pharmaceutics-14-00872" class="html-bibr">150</a>]), (<b>b</b>) nanosphere (reprinted from [<a href="#B150-pharmaceutics-14-00872" class="html-bibr">150</a>]), (<b>c</b>) nanoneedle (created with <a href="http://BioRender.com" target="_blank">BioRender.com</a>, accessed on 6 February 2022), and (<b>d</b>) nanofiber (created with <a href="http://BioRender.com" target="_blank">BioRender.com</a>, accessed on 6 February 2022).</p> Full article ">Figure 9
<p>(<b>a</b>) Schematic representation of the drug diffusion processes from micro or nanoparticles. The figure shows a typical structure of micro/nanoparticles, with a solid drug nucleus: as the dissolution process proceeds, the solid drug diffuses from (i) the micro/nanoparticles to (ii) the medium. Created with <a href="http://BioRender.com" target="_blank">BioRender.com</a> (accessed 6 February 2022). (<b>b</b>) Representations of swelling, erosion, and dissolution. Reprinted from [<a href="#B159-pharmaceutics-14-00872" class="html-bibr">159</a>].</p> Full article ">Figure 10
<p>Comparison of different profiles of drug release kinetics: (<b>a</b>) Zero-order kinetic, (<b>b</b>) first-order kinetic, (<b>c</b>) Higuchi model, and (<b>d</b>) Korsmeyer–Peppas model. Reprinted from [<a href="#B161-pharmaceutics-14-00872" class="html-bibr">161</a>].</p> Full article ">
17 pages, 6111 KiB  
Article
Assessment of the Extrusion Process and Printability of Suspension-Type Drug-Loaded AffinisolTM Filaments for 3D Printing
by Gloria Mora-Castaño, Mónica Millán-Jiménez, Vicente Linares and Isidoro Caraballo
Pharmaceutics 2022, 14(4), 871; https://doi.org/10.3390/pharmaceutics14040871 - 15 Apr 2022
Cited by 20 | Viewed by 3429
Abstract
Three-dimensional (3D) printing technology enables the design of new drug delivery systems for personalised medicine. Polymers that can be molten are needed to obtain extruded filaments for Fused Deposition Modelling (FDM), one of the most frequently employed techniques for 3D printing. The aim [...] Read more.
Three-dimensional (3D) printing technology enables the design of new drug delivery systems for personalised medicine. Polymers that can be molten are needed to obtain extruded filaments for Fused Deposition Modelling (FDM), one of the most frequently employed techniques for 3D printing. The aim of this work was to evaluate the extrusion process and the physical appearance of filaments made of a hydrophilic polymer and a non-molten model drug. Metformin was used as model drug and Affinisol™ 15LV as the main carrier. Drug-loaded filaments were obtained by using a single-screw extruder and, subsequently, their printability was tested. Blends containing up to a 60% and 50% drug load with 5% and 7.5% of auxiliary excipients, respectively, were successfully extruded. Between the obtained filaments, those containing up to 50% of the drug were suitable for use in FDM 3D printing. The studied parameters, including residence time, flow speed, brittleness, and fractal dimension, reflect a critical point in the extrusion process at between 30–40% drug load. This finding could be essential for understanding the behaviour of filaments containing a non-molten component. Full article
(This article belongs to the Special Issue Additive Manufacturing Approaches to Produce Drug Delivery Systems)
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<p>Graphical representation of the extrusion process parameters and filament parameters versus drug percentage of each filament with 5% or 7.5% auxiliary excipients: (<b>A</b>) residence time; (<b>B</b>) flow speed; (<b>C</b>) Df; (<b>D</b>) brittleness.</p> Full article ">Figure 1 Cont.
<p>Graphical representation of the extrusion process parameters and filament parameters versus drug percentage of each filament with 5% or 7.5% auxiliary excipients: (<b>A</b>) residence time; (<b>B</b>) flow speed; (<b>C</b>) Df; (<b>D</b>) brittleness.</p> Full article ">Figure 2
<p>SEM images, magnification 1000×: (<b>A</b>) 40A; (<b>B</b>) 40B.</p> Full article ">Figure 3
<p>SEM images, magnification 100×, of drug-loaded filaments made with blends containing 5% of both MS and PEG 6000: (<b>A</b>) 10A; (<b>B</b>) 20A; (<b>C</b>) 30A; (<b>D</b>) 40A; (<b>E</b>) 50A; (<b>F</b>) 60A.</p> Full article ">Figure 3 Cont.
<p>SEM images, magnification 100×, of drug-loaded filaments made with blends containing 5% of both MS and PEG 6000: (<b>A</b>) 10A; (<b>B</b>) 20A; (<b>C</b>) 30A; (<b>D</b>) 40A; (<b>E</b>) 50A; (<b>F</b>) 60A.</p> Full article ">Figure 4
<p>SEM images, magnification 100×, of drug-loaded filaments made with blends containing 7.5% of both MS and PEG 6000: (<b>A</b>) 20B; (<b>B</b>) 30B; (<b>C</b>) 40B; (<b>D</b>) 50B.</p> Full article ">Figure 5
<p>X-ray tomography image of filament with 50% MET, 40% AFF, and 5% of both auxiliary excipients.</p> Full article ">Figure 6
<p>(<b>A</b>) Digital design of 3D-printed systems, showing the top solid layer and the perimeter. (<b>B</b>) 3D-printed systems made with a 30A filament. (<b>C</b>) SEM image, magnification 50×, of the internal mesh of a printed system made with a 40A filament.</p> Full article ">Figure 7
<p>DSC thermograms of (<b>A</b>) pure materials, (<b>B</b>) drug-loaded filaments.</p> Full article ">
29 pages, 5549 KiB  
Article
Effect of Solvents, Stabilizers and the Concentration of Stabilizers on the Physical Properties of Poly(d,l-lactide-co-glycolide) Nanoparticles: Encapsulation, In Vitro Release of Indomethacin and Cytotoxicity against HepG2-Cell
by Musaed Alkholief, Mohd Abul Kalam, Md Khalid Anwer and Aws Alshamsan
Pharmaceutics 2022, 14(4), 870; https://doi.org/10.3390/pharmaceutics14040870 - 15 Apr 2022
Cited by 21 | Viewed by 3377
Abstract
A biocompatible, biodegradable and FDA-approved polymer [Poly lactic-co-glycolic acid (PLGA)] was used to prepare the nanoparticles (NPs) to observe the effect of solvents, stabilizers and their concentrations on the physical properties of the PLGA-NPs, following the encapsulation and in vitro release [...] Read more.
A biocompatible, biodegradable and FDA-approved polymer [Poly lactic-co-glycolic acid (PLGA)] was used to prepare the nanoparticles (NPs) to observe the effect of solvents, stabilizers and their concentrations on the physical properties of the PLGA-NPs, following the encapsulation and in vitro release of Indomethacin (IND). PLGA-NPs were prepared by the single-emulsion solvent evaporation technique using dichloromethane (DCM)/chloroform as the organic phase with Polyvinyl-alcohol (PVA)/Polyvinylpyrrolidone (PVP) as stabilizers to encapsulate IND. The effects of different proportions of PVA/PVP with DCM/chloroform on the physiochemical properties (particle size, the polydispersity index, the zeta potential by Malvern Zetasizer and morphology by SEM) of the NPs were investigated. DSC was used to check the physical state, the possible complexation of PLGA with stabilizer(s) and the crystallinity of the encapsulated drug. Stabilizers at all concentrations produced spherical, regular-shaped, smooth-surfaced discrete NPs. Average size of 273.2–563.9 nm was obtained when PVA (stabilizer) with DCM, whereas it ranged from 317.6 to 588.1 nm with chloroform. The particle size was 273.2–563.9 nm when PVP was the stabilizer with DCM, while it was 381.4–466.6 nm with chloroform. The zeta potentials of PVA-stabilized NPs were low and negative (−0.62 mV) while they were comparatively higher and positive for PVP-stabilized NPs (+17.73 mV). Finally, drug-loaded optimal NPs were composed of PLGA (40 mg) and IND (4 mg) in 1 mL DCM/chloroform with PVA/PVP (1–3%), which resulted in sufficient encapsulation (54.94–74.86%) and drug loading (4.99–6.81%). No endothermic peak of PVA/PVP appeared in the optimized formulation, which indicated the amorphous state of IND in the core of the PLGA-NPs. The in vitro release study indicated a sustained release of IND (32.83–52.16%) from the PLGA-NPs till 72 h and primarily followed the Higuchi matrix release kinetics followed by Korsmeyer–Peppas models. The cell proliferation assay clearly established that the organic solvents used to prepare PLGA-NPs had evaporated. The PLGA-NPs did not show any particular toxicity in the HepG2 cells within the dose range of IND (250–500 µg/mL) and at an equivalent concentration of PLGA-NPs (3571.4–7142.7 µg/mL). The cytotoxicity of the hepatotoxic drug (IND) was reduced by its encapsulation into PLGA-NPs. The outcomes of this investigation could be implemented to prepare PLGA-NPs of acceptable properties for the encapsulation of low/high molecular weight drugs. It would be useful for further in vitro and in vivo applications to use this delivery system. Full article
(This article belongs to the Special Issue Nanomedicine Formulations Based on PLGA Nanoparticles for Diagnosis, Monitoring and Treatment of Disease: From Bench to Bedside)
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<p>Particle size and zeta potential distribution curves of PLGA-NPs prepared with: (<b>A</b>) 1% PVA and chloroform; (<b>B</b>) 1% PVA and DCM (<b>C</b>) 1% PVP with chloroform (<b>D</b>) 1% PVP with DCM and (<b>E</b>–<b>H</b>) are their zeta potentials, respectively.</p> Full article ">Figure 2
<p>SEM images of the PLGA-NPs prepared with chloroform as organic solvent with different concentrations of PVA in the aqueous phase. (<b>A</b>) 1%, <span class="html-italic">w/v</span> PVA; (<b>B</b>) 3%, <span class="html-italic">w/v</span> PVA; (<b>C</b>) 6%, <span class="html-italic">w/v</span> PVA and (<b>D</b>) 9%, <span class="html-italic">w/v</span> PVA.</p> Full article ">Figure 3
<p>SEM images of the PLGA-NPs prepared with chloroform as organic solvent with different concentrations of PVP in the aqueous phase. (<b>A</b>) 1%, <span class="html-italic">w/v</span> PVP; (<b>B</b>) 3%, <span class="html-italic">w/v</span> PVP; (<b>C</b>) 6%, <span class="html-italic">w/v</span> PVP and (<b>D</b>) 9%, <span class="html-italic">w/v</span> PVP.</p> Full article ">Figure 4
<p>SEM images of the PLGA-NPs prepared with DCM as organic solvent with different concentrations of PVA in the aqueous phase. (<b>A</b>) 1%, <span class="html-italic">w/v</span> PVA; (<b>B</b>) 3%, <span class="html-italic">w/v</span> PVA; (<b>C</b>) 6%, <span class="html-italic">w/v</span> PVA and (<b>D</b>) 9%, <span class="html-italic">w/v</span> PVA.</p> Full article ">Figure 5
<p>SEM images of the PLGA-NPs prepared with DCM as organic solvent with different concentrations of PVP in the aqueous phase. (<b>A</b>) 1%, <span class="html-italic">w/v</span> PVP; (<b>B</b>) 3%, <span class="html-italic">w/v</span> PVP; (<b>C</b>) 6%, <span class="html-italic">w/v</span> PVP and (<b>D</b>) 9%, <span class="html-italic">w/v</span> PVP.</p> Full article ">Figure 6
<p>FT-IR spectra of pure IND (<b>A</b>), PLGA (<b>B</b>), empty PLGA-NPs (<b>C</b>) and IND-loaded PLGA-NPs-5 (<b>D</b>).</p> Full article ">Figure 7
<p>Relationship between viscosity and shear rate in the aqueous solutions of PVA (<b>A</b>) and PVP (<b>B</b>).</p> Full article ">Figure 8
<p>In vitro release profiles of IND from different PLGA-NPs in PBS (pH 7.4). Prepared with different concentrations of stabilizers, (<b>A</b>) PVA and (<b>B</b>) PVP.</p> Full article ">Figure 9
<p>In vitro release profiles of IND from different PLGA-NPs in acidic pH (1.2). Prepared with different concentrations of stabilizers, (<b>A</b>) PVA and (<b>B</b>) PVP.</p> Full article ">Figure 10
<p>Overlay DSC thermograms of Indomethacin. Indomethacin, IND alone (<b>A</b>); PLGA alone (<b>B</b>); PVA alone (<b>C</b>); PVP alone (<b>D</b>) and PLGA-NPs-5 from Batch-2 (<b>E</b>).</p> Full article ">Figure 11
<p>Cytotoxicity after 24 h, 48 h and 72 h of incubation in HepG2 cells: pure IND (<b>A</b>); empty-PLGA-NPs (<b>B</b>) and IND-PLGA-NPs-5 (<b>C</b>) with IC<sub>50</sub> values (µg/mL).</p> Full article ">Figure 12
<p>Histograms of the percentage cell viability of HepG2 after 24 h, 48 h and 72 h of incubation with varying concentrations (5–1000 µg/mL) of pure IND (<b>A</b>) and its equivalent concentration of empty-PLGA-NPs (<b>B</b>) and IND-loaded PLGA-NPs (<b>C</b>). Data were presented as the mean of three independent experiments with error bars as standard deviation (SD).</p> Full article ">
16 pages, 1767 KiB  
Article
Ovarian Follicular Growth through Intermittent Vaginal Gonadotropin Administration in Diminished Ovarian Reserve Women
by Chao-Chin Hsu, Isabel Hsu, Li-Hsuan Lee, Rosie Hsu, Yuan-Shuo Hsueh, Chih-Ying Lin and Hui Hua Chang
Pharmaceutics 2022, 14(4), 869; https://doi.org/10.3390/pharmaceutics14040869 - 15 Apr 2022
Cited by 2 | Viewed by 2381
Abstract
It is a challenge to obtain enough oocytes during in vitro fertilization (IVF) in women who have a poor ovarian response (POR) in achieving conception. We have adopted the characteristics of the first uterine pass effect, which we pioneered in employing the vaginal [...] Read more.
It is a challenge to obtain enough oocytes during in vitro fertilization (IVF) in women who have a poor ovarian response (POR) in achieving conception. We have adopted the characteristics of the first uterine pass effect, which we pioneered in employing the vaginal administration of gonadotropins in women receiving IVF treatments. In our previous study employing vaginal administration, faster absorption and slower elimination of gonadotropins were demonstrated, and, female subjects presented proper ovarian follicle growth and pregnancy rates. In this study, during 2016–2020, 300 to 675 IU of gonadotropins were administered vaginally every three days in 266 POR women for their controlled ovarian hyperstimulation (COH). The injections were performed with needles angled at 15–30° towards the middle-upper portions of the bilateral vaginal wall, with an injection depth of 1–2 mm. For the COH results, these women, on average, received 3.0 ± 0.9 vaginal injections and a total dose of 1318.4 ± 634.4 IU gonadotropins, resulting in 2.2 ± 1.9 mature oocytes and 1.0 ± 1.2 good embryos. Among these embryos, 0.9 ± 1.0 were transferred to reach a clinical pregnancy rate of 18.1% and a live birth rate of 16.7%. In conclusion, the intermittent vaginal administration of gonadotropins proved to be effective in POR women for their IVF treatments. Full article
(This article belongs to the Special Issue Advances in Vaginal Drug Delivery)
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<p>Schematic expression of the vaginal administration of gonadotropins and the possible transportation routes to the ovary. The black solid arrow indicates the major transportation of medicines through lymphatic and venous circulations. The blank dotted line arrow indicates minor transportation of medicines via the countercurrent exchange through vein-to-artery diffusion or direct diffusion.</p> Full article ">Figure 2
<p>Schematic expression of gonadotropin (Gn) administration. The Gn was given on days 2, 5, and 8 for intermittent vaginal administration, in comparison to conventional abdominal injections that are performed daily starting on menstrual day 2. GnRH (Gn-releasing hormone) antagonist was given when the leading follicle reached 12–14 mm in diameter, which was mostly noted at day 8 of the cycle. hCG: human chorionic gonadotropin.</p> Full article ">Figure 3
<p>Flowchart for women of poor ovarian reserve receiving treatment.</p> Full article ">Figure 4
<p>Levels of (<b>A</b>) FSH, (<b>B</b>) E2, and (<b>C</b>) follicles. All data presented as mean ± SEM. <sup>+</sup> The <span class="html-italic">p</span>-value difference was significant between Group C and Group A. <sup>#</sup> The <span class="html-italic">p</span>-value difference was significant between Group B and Group A. * <span class="html-italic">p</span> &lt; 0.05. Group A: AMH &lt; 0.5 ng/mL; Group B: 0.5 ng/mL ≤ AMH &lt; 1.2 ng/mL; Group C: AMH ≥ 1.2 ng/mL.</p> Full article ">
14 pages, 4740 KiB  
Article
Evaluation of CTB-sLip for Targeting Lung Metastasis of Colorectal Cancer
by Xiaoying Zhang, Wenjing Tang, Haoyu Wen, Ercan Wu, Tianhao Ding, Jie Gu, Zhongwei Lv and Changyou Zhan
Pharmaceutics 2022, 14(4), 868; https://doi.org/10.3390/pharmaceutics14040868 - 15 Apr 2022
Cited by 5 | Viewed by 2517
Abstract
Lung metastasis of colorectal cancer is common in the clinic; however, precise targeting for the diagnosis and therapy purposes of those lung metastases remains challenging. Herein, cholera toxin subunit b (CTB) protein was chemically conjugated on the surface of PEGylated liposomes (CTB-sLip). Both [...] Read more.
Lung metastasis of colorectal cancer is common in the clinic; however, precise targeting for the diagnosis and therapy purposes of those lung metastases remains challenging. Herein, cholera toxin subunit b (CTB) protein was chemically conjugated on the surface of PEGylated liposomes (CTB-sLip). Both human-derived colorectal cancer cell lines, HCT116 and HT-29, demonstrated high binding affinity and cellular uptake with CTB-sLip. In vivo, CTB-sLip exhibited elevated targeting capability to the lung metastasis of colorectal cancer in the model nude mice in comparison to PEGylated liposomes (sLip) without CTB modification. CTB conjugation induced ignorable effects on the interaction between liposomes and plasma proteins but significantly enhanced the uptake of liposomes by numerous blood cells and splenic cells, leading to relatively rapid blood clearance in BALB/c mice. Even though repeated injections of CTB-sLip induced the production of anti-CTB antibodies, our results suggested CTB-sLip as promising nanocarriers for the diagnosis of lung metastasis of colorectal cancer. Full article
(This article belongs to the Special Issue Strategies to Enhance Drug Permeability across Biological Barriers)
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<p>Expression and characterization of the CTB protein. (<b>a</b>) Schematically illustration of the structure of CTB. (<b>b</b>) Characterization of the expressed and refolded CTB by SDS-PAGE. (<b>c</b>) Binding activity of the expressed CTB to GM1; BSA was set as the control. The K<sub>d</sub> value was calculated using GraphPad Prism 8.0.1. Data are means ± SDs, <span class="html-italic">n</span> = 3.</p> Full article ">Figure 2
<p>Preparation and characterization of CTB-conjugated PEGylated liposomes (CTB-sLip). (<b>a</b>) Schematic illustration of the conjugation method of CTB to the maleimide group grafted on the surface of PEGylated liposomes. (<b>b</b>) Size and zeta potential characterization of the prepared CTB-sLip and fluorescence dye DiI-loaded CTB-sLip. The binding activities of CTB-sLip/DiI to GM1 coated (<b>c</b>) and BSA coated (<b>d</b>) were performed by ELISA; sLip/DiI and Mal-sLip/DiI were set as the controls. Data are means ± SDs, <span class="html-italic">n</span> = 3.</p> Full article ">Figure 3
<p>Effects of BALB/c plasma proteins on the bioactivity of CTB-sLip. (<b>a</b>) Affinity characterization of CTB-sLip (pre-incubation with BALB/c serum and PBS for 24 h) to GM1 based on ELISA; sLip were set as a control. Data are means ± SDs, <span class="html-italic">n</span> = 3. (<b>b</b>) Evaluation of protein corona of CTB-sLip (with 0.1‰, 0.2‰ or 0.4‰ molar ratio CTB modification) by SDS-PAGE.</p> Full article ">Figure 4
<p>Effect of BALB/c serum on the targeting capability of CTB-sLip to human-derived colorectal cancer cells. CTB-sLip/FAM (5 μM 5-FAM) were prepared to study the in vitro uptake by HCT116 cell ((<b>a</b>), <span class="html-italic">n</span> = 3) and HT-29 cell ((<b>b</b>), <span class="html-italic">n</span> = 4) at 37 °C after 2 h incubation, in 0.1% BSA and 10% BALB/c serum, respectively. Data are means ± SDs. * <span class="html-italic">p</span> &lt; 0.05 and *** <span class="html-italic">p</span> &lt; 0.001 based on one-way ANOVA with Prism.8.0.1.</p> Full article ">Figure 5
<p>Pharmacokinetic profiles (<b>a</b>) and biodistribution (<b>b</b>) of sLip/DiI and CTB-sLip/DiI in major organs of BALB/c mice over 24 h after intravenous injection with 50 mg kg<sup>−1</sup> lipids. Data are means ± SDs, <span class="html-italic">n</span> = 5. * <span class="html-italic">p</span> &lt; 0.05, *** <span class="html-italic">p</span> &lt; 0.001; ns indicates non-significant based on Student’s <span class="html-italic">t</span>-tests.</p> Full article ">Figure 6
<p>Uptake of CTB-sLip by blood white cells of BALB/c mice in vivo. CTB-sLip/DiO were injected with 50 mg kg<sup>−1</sup> lipids. Uptake by neutrophils and monocytes (<b>a</b>), and T cells and B cells (<b>b</b>) in peripheral blood at 2 h and 24 h after injection was determined by Flow Cytometry. Data are means ± SDs (<span class="html-italic">n</span> = 3). ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001; ns indicates nonsignificant based on grouped two-way ANOVAs.</p> Full article ">Figure 7
<p>Uptake of CTB-sLip/DiO by hepatic cells and splenic cells. (<b>a</b>) Kupffer’s cells and liver sinusoidal endothelial cells (LSECs) were marked with F4/80 and CD146, respectively. (<b>b</b>) Splenic macrophages (MΦ), DC cells, splenic B cells, and T cells were marked with F4/80, CD11c, CD19, and CD3, respectively. Data are means ± SDs (<span class="html-italic">n</span> = 3). * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001; ns represents nonsignificant based on grouped two-way ANOVAs.</p> Full article ">Figure 8
<p>Targeting capability of CTB-sLip to lung metastasis of colorectal cancer in vivo. Male BALB/c nude mice planted with HT-29 cells in left lungs were injected with CTB-sLip/DiR ((<b>a</b>,<b>b</b>), <span class="html-italic">n</span> = 3) for in situ imaging and CTB-sLip/DiO ((<b>c</b>,<b>d</b>), <span class="html-italic">n</span> = 5) for cellular uptake study; sLip/DiR and sLip/DiO were set as the controls. Data are means ± SDs. ** <span class="html-italic">p</span> &lt; 0.01 based on Student’s <span class="html-italic">t</span>-tests.</p> Full article ">Figure 9
<p>Immunogenicity of CTB-sLip in vivo. BALB/c mice were sequentially injected with sLip or CTB-sLip (50 mg kg<sup>−1</sup> lipids) per week for 21 days. Anti-PEG IgG/M (<b>a</b>,<b>b</b>) and anti-CTB IgG/M (<b>c</b>,<b>d</b>) in serum on day 21 were detected by ELISA. Data are means ± SDs (<span class="html-italic">n</span> = 3).</p> Full article ">
19 pages, 765 KiB  
Review
Therapeutic Vaccines Targeting Neoantigens to Induce T-Cell Immunity against Cancers
by Shih-Cheng Pao, Mu-Tzu Chu and Shuen-Iu Hung
Pharmaceutics 2022, 14(4), 867; https://doi.org/10.3390/pharmaceutics14040867 - 15 Apr 2022
Cited by 12 | Viewed by 4155
Abstract
Cancer immunotherapy has achieved multiple clinical benefits and has become an indispensable component of cancer treatment. Targeting tumor-specific antigens, also known as neoantigens, plays a crucial role in cancer immunotherapy. T cells of adaptive immunity that recognize neoantigens, but do not induce unwanted [...] Read more.
Cancer immunotherapy has achieved multiple clinical benefits and has become an indispensable component of cancer treatment. Targeting tumor-specific antigens, also known as neoantigens, plays a crucial role in cancer immunotherapy. T cells of adaptive immunity that recognize neoantigens, but do not induce unwanted off-target effects, have demonstrated high efficacy and low side effects in cancer immunotherapy. Tumor neoantigens derived from accumulated genetic instability can be characterized using emerging technologies, such as high-throughput sequencing, bioinformatics, predictive algorithms, mass-spectrometry analyses, and immunogenicity validation. Neoepitopes with a higher affinity for major histocompatibility complexes can be identified and further applied to the field of cancer vaccines. Therapeutic vaccines composed of tumor lysates or cells and DNA, mRNA, or peptides of neoantigens have revoked adaptive immunity to kill cancer cells in clinical trials. Broad clinical applicability of these therapeutic cancer vaccines has emerged. In this review, we discuss recent progress in neoantigen identification and applications for cancer vaccines and the results of ongoing trials. Full article
(This article belongs to the Special Issue Novel Anticancer Strategies (Volume II))
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<p>Schematic representation of neoantigen selection for therapeutic cancer vaccines. The DNA samples are extracted from cancer tissues and peripheral blood mononuclear cells (PBMC), respectively. Non-synonymous mutations and HLA types are obtained through whole-exome sequencing (WES) and HLA genotyping following bioinformatic analyses. The potential neoepitopes derived from the identified mutations are prioritized according to (1) algorithm prediction or (2) immunopeptidome analysis. Afterward, each prioritized neoepitope is examined by immunological assays (e.g., ELISpot or flow cytometry) to validate the immunogenicity. Vaccines encoding the selected neoepitopes are then generated in various formats, including DNA-based, RNA-based, peptide-based, and dendritic-cell (DC)-based vaccines.</p> Full article ">
48 pages, 3370 KiB  
Review
Emerging Nanotherapeutic Approaches to Overcome Drug Resistance in Cancers with Update on Clinical Trials
by Syed Nasir Abbas Bukhari
Pharmaceutics 2022, 14(4), 866; https://doi.org/10.3390/pharmaceutics14040866 - 15 Apr 2022
Cited by 30 | Viewed by 4658
Abstract
A key issue with modern cancer treatments is the emergence of resistance to conventional chemotherapy and molecularly targeted medicines. Cancer nanotherapeutics were created in order to overcome the inherent limitations of traditional chemotherapeutics. Over the last few decades, cancer nanotherapeutics provided unparalleled opportunities [...] Read more.
A key issue with modern cancer treatments is the emergence of resistance to conventional chemotherapy and molecularly targeted medicines. Cancer nanotherapeutics were created in order to overcome the inherent limitations of traditional chemotherapeutics. Over the last few decades, cancer nanotherapeutics provided unparalleled opportunities to understand and overcome drug resistance through clinical assessment of rationally designed nanoparticulate delivery systems. In this context, various design strategies such as passive targeting, active targeting, nano-drug, and multimodal nano-drug combination therapy provided effective cancer treatment. Even though cancer nanotherapy has made great technological progress, tumor biology complexity and heterogeneity and a lack of comprehensive knowledge of nano-bio interactions remain important roadblocks to future clinical translation and commercialization. The current developments and advancements in cancer nanotherapeutics employing a wide variety of nanomaterial-based platforms to overcome cancer treatment resistance are discussed in this article. There is also a review of various nanotherapeutics-based approaches to cancer therapy, including targeting strategies for the tumor microenvironment and its components, advanced delivery systems for specific targeting of cancer stem cells (CSC), as well as exosomes for delivery strategies, and an update on clinical trials. Finally, challenges and the future perspective of the cancer nanotherapeutics to reverse cancer drug resistance are discussed. Full article
(This article belongs to the Special Issue Targeting Drug Resistance and Metastatic Pathways for Cancer Therapy)
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<p>Different nanotherapeutic approaches for overcoming cancer drug resistance. Reproduced from Ref. [<a href="#B12-pharmaceutics-14-00866" class="html-bibr">12</a>], (2022), with permission from Elsevier.</p> Full article ">Figure 2
<p>Active and passive targeting approaches in cancer nanotherapeutics. Reproduced from Ref. [<a href="#B32-pharmaceutics-14-00866" class="html-bibr">32</a>], (2022), with permission from American Chemical Society (ACS).</p> Full article ">Figure 3
<p>Different underlying mechanisms associated with drug resistance. This figure shows different intrinsic and extrinsic factors responsible for cancer drug resistance starting from alteration of signaling pathways, remodeling of drug efflux pumps expression, overexpression of genes related to cell cycle and apoptosis, enhanced expression of nucleic acid synthesis genes, enhanced DNA repair ability, alteration of drug target sites, alteration in functioning of drug metabolizing enzymes, genetic alternations, and epigenetics. Reproduced from Ref. [<a href="#B40-pharmaceutics-14-00866" class="html-bibr">40</a>], (2022), with permission from Elsevier.</p> Full article ">Figure 4
<p>Cancer nanotherapeutics steps: (<b>A</b>) Nanoparticles with a protective layer loaded with chemotherapeutics of interest and decorated with target specific ligand. The drug loaded in the core of the nanoparticle can specifically recognize target cells using target-specific ligands. (<b>B</b>) Nanotherapeutics can reach primary and secondary tumors after entering the circulatory system and target specific tumor cells through the intravasation and extravasation processes. (<b>C</b>) Encapsulated chemotherapeutics are released after binding of tumor specific ligand and target cells surface receptors that causes cellular death. Reproduced from Ref. [<a href="#B34-pharmaceutics-14-00866" class="html-bibr">34</a>], (2022), with permission from Elsevier.</p> Full article ">Figure 5
<p>Cellular components of tumor environment targeted by nanoparticulate system for cancer therapy. Reproduced from Ref. [<a href="#B115-pharmaceutics-14-00866" class="html-bibr">115</a>], (2022), with permission from Elsevier.</p> Full article ">Figure 6
<p>Different nanocarriers being utilized for targeting TME to overcome multi-drug resistance: (<b>a</b>) Nanoparticles loaded with two different drugs for co-delivery at target site for synergistic therapeutic action. (<b>b</b>) Theranostic nanoparticles co-loaded with both therapeutic and diagnostic agents. (<b>c</b>) Stimuli responsive nanoparticles respond against different components of TME such as pH change, ions change, different oxygenation. (<b>d</b>) Multifunctional branched polymeric dendrimer-based nanocarrier loaded with drugs. (<b>e</b>) Nanoparticles loaded with both drugs and nucleic acids (siRNA, miRNA) for synergistic therapeutic action (<b>f</b>) Liposomes loaded drugs for targeted delivery. Reproduced from Ref. [<a href="#B24-pharmaceutics-14-00866" class="html-bibr">24</a>], (2022), with permission from Elsevier.</p> Full article ">Figure 7
<p>Cancer nanotherapeutics approaches to counter hypoxic conditions within tumor microenvironment, which is prime contributing factor for drug resistance. In this approach, specific drugs that can counter hypoxic environment are loaded within nanocarriers. Nanoparticulate system further specifically releases drugs in tumor microenvironment to modulate the hypoxic environment and causes cell death. Reproduces from Ref. [<a href="#B119-pharmaceutics-14-00866" class="html-bibr">119</a>], (2022), with permission from Elsevier.</p> Full article ">
22 pages, 7150 KiB  
Article
Bio-Responsive Carriers for Controlled Delivery of Doxorubicin to Cancer Cells
by Gheorghe Fundueanu, Marieta Constantin, Mihaela Turtoi, Sanda-Maria Bucatariu, Bogdan Cosman, Maria Anghelache, Geanina Voicu and Manuela Calin
Pharmaceutics 2022, 14(4), 865; https://doi.org/10.3390/pharmaceutics14040865 - 15 Apr 2022
Cited by 9 | Viewed by 2543
Abstract
The cellular internalization of drug carriers occurs via different endocytic pathways that ultimately involve the endosomes and the lysosomes, organelles where the pH value drops to 6.0 and 5.0, respectively. We aimed to design and characterize pH/temperature-responsive carriers for the effective delivery of [...] Read more.
The cellular internalization of drug carriers occurs via different endocytic pathways that ultimately involve the endosomes and the lysosomes, organelles where the pH value drops to 6.0 and 5.0, respectively. We aimed to design and characterize pH/temperature-responsive carriers for the effective delivery of the anti-tumoral drug doxorubicin. To this purpose, poly(N-isopropylacrylamide-co-vinylimidazole) was synthesized as an attractive pH/temperature-sensitive copolymer. Microspheres made of this copolymer, loaded with doxorubicin (MS-DXR), disintegrate in monodisperse nanospheres (NS-DXR) under conditions similar to that found in the bloodstream (pH = 7.4, temperature of 36 °C) releasing a small amount of payload. However, in environments that simulate the endosomal and lysosomal conditions, nanospheres solubilize, releasing the entire amount of drug. We followed the NS-DXR internalization using two cancer cell lines, hepatic carcinoma HepG2 cells and lung adenocarcinoma A549 cells. The data showed that NS-DXR are internalized to a greater extent by HepG2 cells than A549 cells, and this correlated with increased cytotoxicity induced by NS-DXR in HepG2 cells compared with A549 cells. Moreover, NS-DXR particles do not cause hemolysis and erythrocytes aggregation. Administered in vivo, NS-DXR localized in the liver and kidneys of mice, and the loading of DXR into NS resulted in the reduced renal clearance of DXR. In conclusion, the newly developed poly(N-isopropylacrylamide-co-vinyl imidazole) particles are biocompatible and may be introduced as carriers for doxorubicin to hepatic tumors. Full article
(This article belongs to the Special Issue Controlled Release of Nanostructured Drug Systems (Volume II))
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<p>Characterization of poly(NIPAAm-co-VI) copolymers: <sup>1</sup>H-NMR spectra in D<sub>2</sub>O (<b>A</b>); potentiometric titration curves of polymer aqueous solution (sample VI<sub>2</sub> in <a href="#pharmaceutics-14-00865-t001" class="html-table">Table 1</a>) in the absence (empty symbols) and in the presence (filled symbols) of 0.1 M NaCl (<b>B</b>); dependence of p<span class="html-italic">K</span><sub>a,</sub><sup>app</sup> on the dissociation degree <span class="html-italic">α</span> (<b>C</b>).</p> Full article ">Figure 2
<p>Particle characterization: scanning electron micrographs of DXR-loaded microspheres obtained by w/o solvent evaporation method (<b>A</b>,<b>B</b>); size distribution of microspheres (<b>C</b>); size distribution of NS-DXR in PBS (pH = 7.4) at 36 °C (<b>D</b>). TEM image of NS-DXR (<b>E</b>). In vitro cumulative release (%) of DXR from NS-DXR under physiological conditions (PB) at pH = 7.4 (circles) and under slightly acidic conditions at pH = 6.6 (diamonds), 6.0 (triangles), 5.5 (empty circles), and 5.0 (squares) (<b>F</b>). Where statistical error bars are not shown, they are smaller than the symbols. Results are expressed as means ± standard deviation (S.D.).</p> Full article ">Figure 3
<p>The viability of HepG2 (<b>A</b>) and A549 (<b>B</b>) cells after exposure to plain NS, NS-DXR, and free DXR determined by XTT assay. The cytotoxicity of plain NS, NS-DXR, and free DXR on HepG2 (<b>C</b>) and A549 (<b>D</b>) cells determined by ToxiLight<sup>TM</sup> assay. Cells were exposed for 24 h to various concentrations of NS and NS-DXR (3.9 ÷125 μg/mL, C<sub>NS</sub> = 125 μg/mL) and the corresponding concentrations of free DXR (0.078 ÷ 2.5 µg/mL, C<sub>DXR</sub> = 2.5 μg/mL). Results are expressed as means ± standard deviation (S.D.) of three experiments performed at least in triplicates. * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.00, **** <span class="html-italic">p</span> &lt; 0.0001 vs. control; # <span class="html-italic">p</span> &lt; 0.05, ## <span class="html-italic">p</span> &lt; 0.01, ### <span class="html-italic">p</span> &lt; 0.001 vs. free DXR.</p> Full article ">Figure 4
<p>DXR fluorescence visualized in HepG2 (<b>A</b>) and A549 cells (<b>C</b>) incubated with NS-DXR and free DXR for 6 and 24 h. Cells were exposed to two doses of DXR either free or loaded into NS-DXR (1.25 and 2.5 µg/mL). The nanoparticle concentrations corresponding to 1.25 and 2.5 μg/mL of loaded DXR are 62.5 and 125 μg/mL, respectively. Representative fluorescence microscopy images show the internalized DXR (red), nuclei stained by DAPI (blue) and F-actin, obtained by cell staining with FITC-labeled phalloidin (green). Merged-1 are the merged images of DXR and nuclei, whereas Merged-2 are the merged images of DXR, nuclei, and F-actin. Scale bar: 50 μm. Quantification of NS-DXR and DXR uptake by HepG2 cells (<b>B</b>) and A549 cells (<b>D</b>) expressed as the ratio of red fluorescence to nuclei number. Each point represents a mean of 27 fields. The bar graph shows results expressed as means ± standard deviation (S.D.) of three experiments performed in triplicate. Statistical significance: *** <span class="html-italic">p</span> &lt; 0.001, # <span class="html-italic">p</span> &lt; 0.05.</p> Full article ">Figure 4 Cont.
<p>DXR fluorescence visualized in HepG2 (<b>A</b>) and A549 cells (<b>C</b>) incubated with NS-DXR and free DXR for 6 and 24 h. Cells were exposed to two doses of DXR either free or loaded into NS-DXR (1.25 and 2.5 µg/mL). The nanoparticle concentrations corresponding to 1.25 and 2.5 μg/mL of loaded DXR are 62.5 and 125 μg/mL, respectively. Representative fluorescence microscopy images show the internalized DXR (red), nuclei stained by DAPI (blue) and F-actin, obtained by cell staining with FITC-labeled phalloidin (green). Merged-1 are the merged images of DXR and nuclei, whereas Merged-2 are the merged images of DXR, nuclei, and F-actin. Scale bar: 50 μm. Quantification of NS-DXR and DXR uptake by HepG2 cells (<b>B</b>) and A549 cells (<b>D</b>) expressed as the ratio of red fluorescence to nuclei number. Each point represents a mean of 27 fields. The bar graph shows results expressed as means ± standard deviation (S.D.) of three experiments performed in triplicate. Statistical significance: *** <span class="html-italic">p</span> &lt; 0.001, # <span class="html-italic">p</span> &lt; 0.05.</p> Full article ">Figure 5
<p>In vitro hemocompatibility evaluation of NS, NS-DXR, and DXR. (<b>A</b>) Photographs of the sedimented erythrocytes after their exposure to various concentrations of NS and NS-DXR (0.0078 ÷ 1 mg/mL, C<sub>NS</sub> = 1 mg/mL), and to the corresponding free DXR concentrations (0.156 ÷ 20 μg/mL, C<sub>DXR</sub> = 20 µg/mL). (<b>B</b>) Quantification of hemolysis by measuring the absorbance of hemoglobin at 540 nm. Results are expressed as means ± standard deviation (S.D.) of one experiment performed in duplicate.</p> Full article ">Figure 6
<p>(<b>A</b>) Localization of NS-DXR and free DXR in organs harvested from C57BL/6 mice. The analysis was completed 1 h after the retro-orbital injection of NS-DXR (n = 4), free DXR (n = 3), or PBS (n = 2) in mice using the imaging system IVIS Caliper 200, by detection of the DXR fluorescence using the filter set λ<sub>ex</sub>/λ<sub>em</sub>: 500 nm/600 nm. (<b>B</b>) Quantification of total radiant efficiency in organs using the region-of-interest option of Living Image software; the resulting intensities were reported to the corresponding organ weight (g). (<b>C</b>) Spectral unmixing analysis to delineate the signal specific for DXR fluorescence and tissue autofluorescence. The composite image displays DXR fluorescence (red) and tissue autofluorescence (green). (<b>D</b>) Quantification of DXR in plasma and urine of mice, 1 h after administration of 3 mg/kg of free DXR or incorporated into NS-DXR, at λ<sub>ex</sub> = 480 nm and λ<sub>em</sub> = 590 nm. * <span class="html-italic">p</span> &lt; 0.05, *** <span class="html-italic">p</span> &lt; 0.001. (<b>E</b>) Evaluation of in vivo hemocompatibility by following the erythrocyte aggregation after the administration of NS-DXR and free DXR. Samples from mice who received PBS were considered the negative control. Scale bar: 50 μm.</p> Full article ">Figure 7
<p>Schematic representation of the potential behavior of DXR-loaded microspheres (MS-DXR) made of poly(N-isopropylacrylamide-co-vinylimidazole) copolymer after suspension in physiological buffers and interaction with a tumor cell. The synthesized MS-DXR (<b>A</b>) disintegrate in monodisperse stable nanoparticles (NS-DXR) in conditions simulating the bloodstream (PBS, pH = 7.4, T = 36 °C) (<b>B</b>), that are internalized by the cancer cells by endocytosis (<b>C</b>). After internalization, the NS-DXR arrives in the late endosomes (pH: 6.0–5.0), where they start to disintegrate and release the drug cargo.</p> Full article ">
21 pages, 1502 KiB  
Article
Chiral Recognition R- and RS- of New Antifungal: Complexation/Solubilization/Dissolution Thermodynamics and Permeability Assay
by Tatyana V. Volkova, Olga R. Simonova, Igor B. Levshin and German L. Perlovich
Pharmaceutics 2022, 14(4), 864; https://doi.org/10.3390/pharmaceutics14040864 - 15 Apr 2022
Cited by 4 | Viewed by 1826
Abstract
Novel potential antifungal of 1,2,4-triazole class have been synthesized as pure enantiomer (R-98) and racemic (RS-186). The effect of 2-hydroxypropyl-β-cyclodextrin (CD) on the solubility and permeability of RS-186 and R-98 in terms of chiral recognition was investigated. Phase solubility studies were [...] Read more.
Novel potential antifungal of 1,2,4-triazole class have been synthesized as pure enantiomer (R-98) and racemic (RS-186). The effect of 2-hydroxypropyl-β-cyclodextrin (CD) on the solubility and permeability of RS-186 and R-98 in terms of chiral recognition was investigated. Phase solubility studies were carried out at 4 temperatures in 0–0.05 M CD concentration range for pH 2.0 and pH 7.4. AL- and AL−-type phase-solubility profiles were obtained for both compounds in pH 2.0 and pH 7.4. The racemic formed more stable complexes with CD as compared to R-isomer. Disclosing of chiral discrimination was facilitated using the approach based on the complex consideration of the derived complexation/solubilization/inherent dissolution thermodynamic functions, including the differential parameters between the racemic compound and R-enantiomer. The differences in the thermodynamic parameters determined by the chirality were discussed in terms of the driving forces of the processes and the main interactions of the compounds with CD in solution. The membrane permeability of both samples in the presence of CD was accessed in order to evaluate the specificity of enantioselective transport through the lipophilic membrane. The solubility/permeability interrelation was disclosed. The investigated compounds were classified as medium permeable in pure buffers and low permeable in the presence of 0.01 M CD. The obtained results can be useful for the design of pharmaceutical products in the form of liquid formulations based on the investigated substances. Full article
(This article belongs to the Special Issue Cyclodextrins and Their Inclusion Complexes for Pharmaceutical Uses)
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Graphical abstract

Graphical abstract
Full article ">Figure 1
<p>Structure of the studied compounds. Chiral centers are marked by *.</p> Full article ">Figure 2
<p>The absorption spectra of RS-186 in pure buffer (1), with 0.05 M 2-HP-β-<span class="html-italic">CD</span> (2) and n-hexane (3); pH 2.0—(<b>a</b>), pH 7.4—(<b>b</b>). Solid lines mean the spectra of pure solvents; dashed lines indicate the spectra of aqueous 2-HP-β-<span class="html-italic">CD</span> solutions.</p> Full article ">Figure 2 Cont.
<p>The absorption spectra of RS-186 in pure buffer (1), with 0.05 M 2-HP-β-<span class="html-italic">CD</span> (2) and n-hexane (3); pH 2.0—(<b>a</b>), pH 7.4—(<b>b</b>). Solid lines mean the spectra of pure solvents; dashed lines indicate the spectra of aqueous 2-HP-β-<span class="html-italic">CD</span> solutions.</p> Full article ">Figure 3
<p>Correlation between the free energy of complex formation (<math display="inline"><semantics> <mrow> <mo>Δ</mo> <msubsup> <mi>G</mi> <mi>C</mi> <mrow> <mi>T</mi> <mo>,</mo> <mi>X</mi> </mrow> </msubsup> </mrow> </semantics></math>) and free energy of the inherent solubility (<math display="inline"><semantics> <mrow> <mo>Δ</mo> <msubsup> <mi>G</mi> <mrow> <mi>s</mi> <mi>o</mi> <mi>l</mi> </mrow> <mrow> <mi>T</mi> <mo>,</mo> <mi>X</mi> </mrow> </msubsup> </mrow> </semantics></math>) for R-98 (black opened squares) and RS-186 (black opened circles) at pH 2.0 (mole fraction scale).</p> Full article ">Figure 4
<p>Solubility-permeability interrelation exemplified by the effect of <span class="html-italic">CD</span> concentration on R-98: apparent permeability coefficient—black opened squares—□, solubility—black filled squares—■; and R-186: permeability coefficient—black opened circles—○, solubility—black filled circles—●. (<b>a</b>) pH 2.0; (<b>b</b>) pH 7.4. The solid and dashed lines indicate the solubility and permeability dependences, respectively.</p> Full article ">
18 pages, 2154 KiB  
Article
Fabrication and In Vitro/In Vivo Appraisal of Metronidazole Intra-Gastric Buoyant Sustained-Release Tablets in Healthy Volunteers
by Mohammed H. Elkomy, Heba A. Abou-Taleb, Hussein M. Eid and Heba A. Yassin
Pharmaceutics 2022, 14(4), 863; https://doi.org/10.3390/pharmaceutics14040863 - 14 Apr 2022
Cited by 9 | Viewed by 2193
Abstract
Helicobacter pylori is thought to be the most common cause of peptic and duodenal ulcers. Eradication of this organism is now considered one of the lines of treatment of gastric and duodenal ulcers. This can be achieved via local delivery of antibacterial agents [...] Read more.
Helicobacter pylori is thought to be the most common cause of peptic and duodenal ulcers. Eradication of this organism is now considered one of the lines of treatment of gastric and duodenal ulcers. This can be achieved via local delivery of antibacterial agents in high concentrations. Accordingly, our objective was to fabricate and evaluate sustained release floating tablets for metronidazole to extend the gastric residence period and control the release rate of metronidazole. Floating tablets containing cellulose derivatives and Avicel were prepared using direct compression. The rate of metronidazole release from the floating tablets (K = 6.278 mg min−1/2) was significantly lower than that from conventional tablets (K = 10.666 mg min−1/2), indicating sustained drug release, according to the Higuchi model, for more than 6 h in an acidic medium of 0.1 N HCl. In vivo study in healthy volunteers revealed significantly improved bioavailability; increased Tmax, AUC, and MRT; and significantly lower absorption rate constant after a single oral dose of 150 mg metronidazole as floating tablets. In addition, the significant increase in MRT indicated an in vivo sustained drug release. The floating tablets provided several benefits, including ease of preparation, absence of effervescent ingredients, and reliance on a pH-independent gel-forming agent to deliver metronidazole in a sustained manner. In conclusion, the prepared tablets could be promising for enhancing both local and systemic metronidazole efficacy. Full article
(This article belongs to the Section Pharmaceutical Technology, Manufacturing and Devices)
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<p>Chemical structure of metronidazole.</p> Full article ">Figure 2
<p>Effect of metronidazole content on the floating features of metronidazole tablets (800 mg).</p> Full article ">Figure 3
<p>Effect of metronidazole content in 800 mg HPMC tablets on the apparent density and maximum hardness of immediate buoyancy (MHIB).</p> Full article ">Figure 4
<p>Percentage metronidazole released versus time profiles of buoyant and conventional tablets in 0.1 N HCl at 37 °C.</p> Full article ">Figure 5
<p>Standard curve of metronidazole (<span class="html-italic">n</span> = 3).</p> Full article ">Figure 6
<p>Representative HPLC chromatograms for metronidazole-free blank serum sample (<b>A</b>), serum sample spiked with metronidazole (1 µg/mL) (<b>B</b>), and serum sample obtained 6 h after metronidazole administration (<b>C</b>).</p> Full article ">Figure 7
<p>Average metronidazole serum concentration–time profiles in human volunteers following oral administration of a single dose of 150 mg as floating and conventional tablets (<span class="html-italic">n</span> = 6).</p> Full article ">Figure 8
<p>Percentage coefficient of variation of metronidazole serum concentration in conventional and floating tablets.</p> Full article ">
17 pages, 2829 KiB  
Article
Squalene-Based Nano-Assemblies Improve the Pro-Autophagic Activity of Trehalose
by Giulia Frapporti, Eleonora Colombo, Hazem Ahmed, Giulia Assoni, Laura Polito, Pietro Randazzo, Daniela Arosio, Pierfausto Seneci and Giovanni Piccoli
Pharmaceutics 2022, 14(4), 862; https://doi.org/10.3390/pharmaceutics14040862 - 14 Apr 2022
Cited by 9 | Viewed by 2471
Abstract
The disaccharide trehalose is a well-established autophagy inducer, but its therapeutic application is severely hampered by its low potency and poor pharmacokinetic profile. Thus, we targeted the rational design and synthesis of trehalose-based small molecules and nano objects to overcome such issues. Among [...] Read more.
The disaccharide trehalose is a well-established autophagy inducer, but its therapeutic application is severely hampered by its low potency and poor pharmacokinetic profile. Thus, we targeted the rational design and synthesis of trehalose-based small molecules and nano objects to overcome such issues. Among several rationally designed trehalose-centered putative autophagy inducers, we coupled trehalose via suitable spacers with known self-assembly inducer squalene to yield two nanolipid-trehalose conjugates. Squalene is known for its propensity, once linked to a bioactive compound, to assemble in aqueous media in controlled conditions, internalizing its payload and forming nanoassemblies with better pharmacokinetics. We assembled squalene conjugates to produce the corresponding nanoassemblies, characterized by a hydrodynamic diameter of 188 and 184 nm and a high stability in aqueous media as demonstrated by the measured Z-potential. Moreover, the nanoassemblies were characterized for their toxicity and capability to induce autophagy in vitro. Full article
(This article belongs to the Special Issue Novel Pharmaceutical and Pharmacological Strategies to Induce Autophagy)
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Graphical abstract

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Full article ">Figure 1
<p>Assessment of the cellular toxicity induced by trehalose, conjugates <b>1a</b> and <b>1b</b> and NAs <b>5a</b> and <b>5b</b>. We evaluated cell viability upon compound treatment by MTT. (<b>A</b>) Cytotoxicity assay on HeLa cells treated for 24 h with trehalose, squalene, 4,4’-dithiodibutyric acid (linker), <b>1b</b> and <b>5b</b>. Data are presented as mean ± SE; *** <span class="html-italic">p</span> &lt; 0.001, ** <span class="html-italic">p</span> &lt; 0.01, * <span class="html-italic">p</span> &lt; 0.05 vs. trehalose, <span class="html-italic">n</span> = 10–15. (<b>B</b>) Cytotoxicity assay on HeLa cells treated for 24 h with trehalose, squalene, linker, <b>1a</b> and <b>5a</b>. Data are presented as mean ± SE * <span class="html-italic">p</span> &lt; 0.05 vs. trehalose, <span class="html-italic">n</span> = 10–15.</p> Full article ">Figure 2
<p>Biological activity of trehalose, conjugates <b>1a</b> and <b>1b</b> and NAs <b>5a</b> and <b>5b</b>. We assessed LC3B conversion upon compound treatment by Western blotting. (<b>A</b>) Evaluation of autophagy induction in HeLa cells treated for 24 h with trehalose, <b>1b</b> and <b>1a</b>. Upon treatment, we analyzed tubulin, LC3BI and II levels by Western blotting. (<b>B</b>) Evaluation of autophagy induction in HeLa cells treated for 24 h with trehalose and <b>5b</b>. Upon treatment, we analyzed α-tubulin, LC3BI and II levels by Western blotting. (<b>C</b>) Evaluation of autophagy induction in HeLa cells treated for 24 h with trehalose and <b>5a</b>. Upon treatment, we analyzed α-tubulin, LC3BI and II levels by Western blotting.</p> Full article ">Figure 3
<p>Biological activity of trehalose, conjugates <b>1a</b> and <b>1b</b> and NAs <b>5a</b> and <b>5b</b>. Quantification of LC3BI, LC3BII and tubulin signals. (<b>A</b>) The graph shows LC3BII amount expressed as fold over α-tubulin. Data are expressed as mean ± S.E.; * &lt; <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01 vs. control, <span class="html-italic">n</span> = 10–15. (<b>B</b>) The graph shows the LC3BII/LC3BI ratio. Each value was normalized on proper control. Data are expressed as mean ± S.E.; *** <span class="html-italic">p</span> &lt; 0.001, ** <span class="html-italic">p</span> &lt; 0.01, * <span class="html-italic">p</span> &lt; 0.05 vs. control, <span class="html-italic">n</span> = 10–15.</p> Full article ">Figure 4
<p>Characterization of the effects of trehalose and NAs <b>5a</b> and <b>5b</b> on autophagy. We assessed LC3B conversion upon compound treatment by Western blotting. (<b>A</b>) Evaluation of autophagy induction in HeLa cells treated for 24 h with 25 μM trehalose, 25 μM <b>5a</b> and 10 μM <b>5b</b>. During the last 2 h of treatment, we added the known lysosomal blocker bafilomycin (100 nM, 2 h). Upon treatment, we analyzed tubulin and LC3BII levels by Western blotting. (<b>B</b>) The graph shows LC3BII amounts expressed as fold over α-tubulin. Data are expressed as mean ± S.E.; ** <span class="html-italic">p</span> &lt; 0.01 vs. control, # <span class="html-italic">p</span> &lt; 0.05 vs. control + bafilomycin, <span class="html-italic">n</span> = 8–10.</p> Full article ">Figure 5
<p>Assessment of autophagy by imaging approaches. We investigated the autophagic flux by a GFP-mCherry-LC3B reporter. Non-acidic autophagosomes appear as green and red dots, while autophagolysomes are red. (<b>A</b>) Image-based investigation of autophagy upon expression of the reporter GFP-mCherry-LC3B in HeLa cells treated for 24 h with trehalose (100 mM), <b>5b</b> (10 μM) and <b>5a</b> (25 μM). Scale bar = 10 μm. (<b>B</b>) The graph reports the number of autophagosomes (yellow) and autophagolysomes (red) present in each cell. Data are expressed as mean ± S.E.; * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01 vs. control, <span class="html-italic">n</span> = 10–15 for each experiment, 3 independent experiments. (<b>C</b>) The graph reports the percentage of autophagosomes (yellow) and autophagolysomes (red) present in each cell. Data are expressed as mean ± S.E.; * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01 vs. control, <span class="html-italic">n</span> = 10–15 for each experiment, 3 independent experiments.</p> Full article ">Figure 6
<p>The activity of NAs <b>5a</b> and <b>5b</b> is reduction-dependent in cells. We assessed the role of intracellular glutathione on NA activity by Western blotting. (<b>A</b>) In eukaryotic cells, de novo synthesis of reduced glutathione (GSH) occurs via a two-step ATP-requiring enzymatic process mediated by two different enzymes, γ-glutamylcysteine synthetase and glutathione synthase. Buthionine sulphoximine (BSO) is an inhibitor of gamma-glutamylcysteine synthetase and, consequently, lowers cellular GSH concentrations. (<b>B</b>) Cytotoxicity assay on HeLa cells treated for 24 h with BSO (1 mM) alone, or in combination with <b>5b</b> (10 μM) or <b>5a</b> (25 μM). Data are presented as mean ± SE; <span class="html-italic">n</span> = 10–15. (<b>C</b>) Evaluation of autophagy induction in HeLa cells treated for 24 h with trehalose (100 mM), BSO (1 mM), <b>5a</b> (25 μM), <b>5b</b> (10 μM), BSO (1 mM) + <b>5a</b> (25 μM) and BSO (1 mM) + <b>5b</b> (10 μM). Upon treatment, we analyzed α-tubulin, LC3BI and II levels by Western blotting.</p> Full article ">Figure 7
<p>The activity of NAs <b>5a</b> and <b>5b</b> is reduction-dependent in cells. Quantification of LC3BI, LC3BII and tubulin signal. (<b>A</b>) The graph shows the LC3BII amount expressed as fold over α-tubulin. Data are expressed as mean ± S.E.; * &lt; <span class="html-italic">p</span> &lt; 0.05, vs. compound alone, <span class="html-italic">n</span> = 10–15. (<b>B</b>) The graph shows the LC3BII/LC3BI ratio. Each value was normalized on proper control. Data are expressed as mean ± S.E.; * &lt; <span class="html-italic">p</span> &lt; 0.05vs. compound alone, <span class="html-italic">n</span> = 10–15.</p> Full article ">Scheme 1
<p>Synthesis of mono- (<b>1a</b>) and bis- (<b>1b</b>) squalene-trehalose adducts for self-assembling organic nanovectors.</p> Full article ">Scheme 2
<p>Preparation of 1:1 squalene-trehalose-based (<b>5a</b>, left) and 2:1 (<b>5b</b>, right) squalene-trehalose-based NAs.</p> Full article ">
11 pages, 1745 KiB  
Article
Using a State-Bounding Observer to Predict the Guaranteed Limits of Drug Amounts in Rats after Oral Administration Based on an Uncertain Pharmacokinetic Model
by Zuzana Vitková, Martin Dodek, Jarmila Pavlovičová and Anton Vitko
Pharmaceutics 2022, 14(4), 861; https://doi.org/10.3390/pharmaceutics14040861 - 14 Apr 2022
Cited by 2 | Viewed by 1454
Abstract
In the first part of this paper, the problem of using an uncertain pharmacokinetic model is resolved to determine drug concentrations in rats after the oral administration of drug suspensions with and without added tenside. To this end, a generalized pharmacokinetic model determining [...] Read more.
In the first part of this paper, the problem of using an uncertain pharmacokinetic model is resolved to determine drug concentrations in rats after the oral administration of drug suspensions with and without added tenside. To this end, a generalized pharmacokinetic model determining the guaranteed limits of drug concentrations was designed. Based on this, the design of the so-called state-bounding observer is described in the second part. Rather than being driven by the output of the pharmacokinetic model, the observer can be driven exclusively by a concentration collected from a suitable part of the body and predict the possible risk of the drug concentration not remaining within the therapeutic range for a sufficiently long time. Specifically, the observer determines the upper and lower limits of the concentrations in all the compartments, especially those that are inaccessible for the collection of samples. The proposed approaches are demonstrated by examples. Full article
(This article belongs to the Special Issue Pharmacokinetics of Orally Administered Drugs)
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Figure 1
<p>Structure of the two-compartment model of instantaneous oral administration [<a href="#B1-pharmaceutics-14-00861" class="html-bibr">1</a>].</p> Full article ">Figure 2
<p>Regions in which amounts <math display="inline"><semantics> <mrow> <msub> <mi>x</mi> <mn>1</mn> </msub> </mrow> </semantics></math> (<b>a</b>) and <math display="inline"><semantics> <mrow> <msub> <mi>x</mi> <mn>2</mn> </msub> </mrow> </semantics></math> (<b>b</b>) can vary is delineated by the amount <math display="inline"><semantics> <mrow> <msub> <mi mathvariant="bold-italic">x</mi> <mi>U</mi> </msub> </mrow> </semantics></math> and <math display="inline"><semantics> <mrow> <msub> <mi mathvariant="bold-italic">x</mi> <mi>L</mi> </msub> </mrow> </semantics></math> of the drug without tenside.</p> Full article ">Figure 3
<p>Regions in which amounts <math display="inline"><semantics> <mrow> <msub> <mi>x</mi> <mn>1</mn> </msub> </mrow> </semantics></math> (<b>a</b>) and <math display="inline"><semantics> <mrow> <msub> <mi>x</mi> <mn>2</mn> </msub> </mrow> </semantics></math> (<b>b</b>) can vary is delineated by the amount <math display="inline"><semantics> <mrow> <msub> <mi>x</mi> <mi>U</mi> </msub> </mrow> </semantics></math> and <math display="inline"><semantics> <mrow> <msub> <mi>x</mi> <mi>L</mi> </msub> </mrow> </semantics></math> of the drug with tenside.</p> Full article ">Figure 4
<p>Three-compartment model of drug disposition.</p> Full article ">Figure 5
<p>Practical use of the state-bounding observer.</p> Full article ">Figure 6
<p>Regions in which the amounts <math display="inline"><semantics> <mrow> <msub> <mi>x</mi> <mn>1</mn> </msub> <mo> </mo> </mrow> </semantics></math>(<b>a</b>), <math display="inline"><semantics> <mrow> <msub> <mi>x</mi> <mrow> <mn>2</mn> <mo> </mo> </mrow> </msub> </mrow> </semantics></math> (<b>b</b>), and <math display="inline"><semantics> <mrow> <msub> <mi>x</mi> <mn>3</mn> </msub> <mo> </mo> </mrow> </semantics></math> (<b>c</b>) may potentially vary is delineated by the corresponding curves <math display="inline"><semantics> <mrow> <msub> <mi mathvariant="bold-italic">x</mi> <mi>U</mi> </msub> </mrow> </semantics></math> and <math display="inline"><semantics> <mrow> <msub> <mi mathvariant="bold-italic">x</mi> <mi>L</mi> </msub> </mrow> </semantics></math>.</p> Full article ">
13 pages, 1049 KiB  
Article
Pharmacokinetics of Tildipirosin in Plasma, Milk, and Somatic Cells Following Intravenous, Intramuscular, and Subcutaneous Administration in Dairy Goats
by Juan Sebastián Galecio, Pedro Marín, Verónica Hernandis, María Botía and Elisa Escudero
Pharmaceutics 2022, 14(4), 860; https://doi.org/10.3390/pharmaceutics14040860 - 13 Apr 2022
Cited by 4 | Viewed by 2115
Abstract
Tildipirosin is a macrolide currently authorized for treating respiratory diseases in cattle and swine. The disposition kinetics of tildipirosin in plasma, milk, and somatic cells were investigated in dairy goats. Tildipirosin was administered at a single dose of 2 mg/kg by intravenous (IV) [...] Read more.
Tildipirosin is a macrolide currently authorized for treating respiratory diseases in cattle and swine. The disposition kinetics of tildipirosin in plasma, milk, and somatic cells were investigated in dairy goats. Tildipirosin was administered at a single dose of 2 mg/kg by intravenous (IV) and 4 mg/kg by intramuscular (IM) and subcutaneous (SC) routes. Concentrations of tildipirosin were determined by an HPLC method with UV detection. Pharmacokinetic parameters were estimated by non-compartmental analysis. Muscle damage, cardiotoxicity, and inflammation were evaluated. After IV administration, the apparent volume of distribution in the steady state was 7.2 L/kg and clearance 0.64 L/h/kg. Plasma and milk half-lives were 6.2 and 58.3 h, respectively, indicating nine times longer persistence of tildipirosin in milk than in plasma. Moreover, if somatic cells are considered, persistence and exposure measured by the area under concentration–time curve (AUC) significantly exceeded those obtained in plasma. Similarly, longer half-lives in whole milk and somatic cells compared to plasma were observed after IM and SC administration. No adverse effects were observed. In brief, tildipirosin should be reserved for cases where other suitable antibiotics have been unsuccessful, discarding milk production of treated animals for at least 45 days or treating goats at the dry-off period. Full article
(This article belongs to the Special Issue Specific Drug Disposition in Veterinary Medicine)
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Figure 1
<p>Chromatogram of tildipirosin and IS in a somatic cell experimental sample by HPLC-UV.</p> Full article ">Figure 2
<p>Semilogarithmic plots of intracellular somatic cell, milk, and plasma tildipirosin concentrations in goats after intravenous (<b>A</b>), subcutaneous (<b>B</b>), and intramuscular (<b>C</b>) administration. Values are the arithmetic mean ± CI 95 (<span class="html-italic">n</span> = 6).</p> Full article ">Figure 2 Cont.
<p>Semilogarithmic plots of intracellular somatic cell, milk, and plasma tildipirosin concentrations in goats after intravenous (<b>A</b>), subcutaneous (<b>B</b>), and intramuscular (<b>C</b>) administration. Values are the arithmetic mean ± CI 95 (<span class="html-italic">n</span> = 6).</p> Full article ">
23 pages, 4536 KiB  
Article
Puzzle out Machine Learning Model-Explaining Disintegration Process in ODTs
by Jakub Szlęk, Mohammad Hassan Khalid, Adam Pacławski, Natalia Czub and Aleksander Mendyk
Pharmaceutics 2022, 14(4), 859; https://doi.org/10.3390/pharmaceutics14040859 - 13 Apr 2022
Cited by 11 | Viewed by 3256
Abstract
Tablets are the most common dosage form of pharmaceutical products. While tablets represent the majority of marketed pharmaceutical products, there remain a significant number of patients who find it difficult to swallow conventional tablets. Such difficulties lead to reduced patient compliance. Orally disintegrating [...] Read more.
Tablets are the most common dosage form of pharmaceutical products. While tablets represent the majority of marketed pharmaceutical products, there remain a significant number of patients who find it difficult to swallow conventional tablets. Such difficulties lead to reduced patient compliance. Orally disintegrating tablets (ODT), sometimes called oral dispersible tablets, are the dosage form of choice for patients with swallowing difficulties. ODTs are defined as a solid dosage form for rapid disintegration prior to swallowing. The disintegration time, therefore, is one of the most important and optimizable critical quality attributes (CQAs) for ODTs. Current strategies to optimize ODT disintegration times are based on a conventional trial-and-error method whereby a small number of samples are used as proxies for the compliance of whole batches. We present an alternative machine learning approach to optimize the disintegration time based on a wide variety of machine learning (ML) models through the H2O AutoML platform. ML models are presented with inputs from a database originally presented by Han et al., which was enhanced and curated to include chemical descriptors representing active pharmaceutical ingredient (API) characteristics. A deep learning model with a 10-fold cross-validation NRMSE of 8.1% and an R2 of 0.84 was obtained. The critical parameters influencing the disintegration of the directly compressed ODTs were ascertained using the SHAP method to explain ML model predictions. A reusable, open-source tool, the ODT calculator, is now available at Heroku platform. Full article
(This article belongs to the Special Issue Computational Intelligence (CI) Tools in Drug Discovery and Design)
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Graphical abstract

Graphical abstract
Full article ">Figure 1
<p>Schematic representation of the applied workflow. Models: distributed random forest (DRF), extremely randomized trees (XRT), generalized linear model (GLM), extreme gradient boosting machine (XGBoost), gradient boosting machine (GBM), deep learning (fully connected multilayer artificial neural network, DL), and stacked ensemble (SE); n_try, number of starting points for probing hyperparameter space; cv, cross-validation; API, active pharmaceutical ingredient.</p> Full article ">Figure 2
<p>Correlation matrix of the database.</p> Full article ">Figure 3
<p>Box and violin plot of selected variables present in the database. Boxes represent interquartile range (IQR), namely: first quartile (Q1), median (horizontal line), third quartile (Q3), and the lower whisker = Q1–1.5*IQR; the higher whisker = Q3 + 1.5*IQR; curves represent distributions of numeric data using kernel density function.</p> Full article ">Figure 4
<p>SHAP dependence plot of the top 20 features of the deep learning model. MCC, microcrystalline cellulose; CC-Na, croscarmellose sodium; SSG, sodium starch glycollate; MgSt, magnesium stearate; SSF, sodium stearyl fumarate; API, active pharmaceutical ingredient.</p> Full article ">Figure 5
<p>SHAP plots representing the effects of formulation composition on the disintegration time [s] for: Crosspovidone [%] (<b>A</b>), croscarmellose sodium (CC-Na) [%] (<b>B</b>), sodium starch glycolate (SSG) [%] (<b>C</b>), Eudragit EPO [%] (<b>D</b>), Aerosil [%] (<b>E</b>), Talc [%] (<b>F</b>).</p> Full article ">Figure 6
<p>SHAP plots representing effects of various manufacturing parameters on the disintegration time: punch die of tablet press [mm] (<b>A</b>), thickness [mm] (<b>B</b>), hardness [N] (<b>C</b>), amount of API [%] (<b>D</b>).</p> Full article ">Figure 7
<p>SHAP plots representing the effects of APIs molecular descriptors on disintegration time. GATS7i and GATS7p, the Geary autocorrelation with lag 7 descriptors, weighted by ionization potential (<b>A</b>) or polarizability (<b>B</b>); GGI7, the topological charge index of order 7 (<b>C</b>); MATS4p, the Moran autocorrelation of lag 4 weighted by polarizability (<b>D</b>); MIC2, a modified information content index, neighborhood symmetry of 2-order descriptor (<b>E</b>); XLogP, a theoretical n-octanol–water partition coefficient (<b>F</b>).</p> Full article ">Figure 8
<p>Partial dependency plots for XLogP vs. super-disintegrants: crospovidone (<b>A</b>), CC-Na (<b>B</b>), SSG (<b>C</b>), and lubricants: Aerosil (<b>D</b>), MgSt (<b>E</b>), SSF (<b>F</b>). XLogP, a theoretical n-octanol–water partition coefficient; SSG, sodium starch glycolate; MgSt, magnesium stearate; SSF, sodium stearyl fumarate; CC-Na, croscarmellose sodium.</p> Full article ">
19 pages, 4168 KiB  
Article
Dual Stimuli-Responsive Multifunctional Silicon Nanocarriers for Specifically Targeting Mitochondria in Human Cancer Cells
by Vy Anh Tran, Giau Van Vo, Mario A. Tan, Joon-Seo Park, Seong Soo A. An and Sang-Wha Lee
Pharmaceutics 2022, 14(4), 858; https://doi.org/10.3390/pharmaceutics14040858 - 13 Apr 2022
Cited by 11 | Viewed by 2973
Abstract
Specific targeting, selective stimuli-responsiveness, and controlled release of anticancer agents are requested for high therapeutic efficiency with a minimal adverse effect. Herein, we report the sophisticated synthesis and functionalization of fluorescent mesoporous silicon (FMPSi) nanoparticles decorated with graphene oxide (GO) nanosheets. GO-wrapped FMPSi [...] Read more.
Specific targeting, selective stimuli-responsiveness, and controlled release of anticancer agents are requested for high therapeutic efficiency with a minimal adverse effect. Herein, we report the sophisticated synthesis and functionalization of fluorescent mesoporous silicon (FMPSi) nanoparticles decorated with graphene oxide (GO) nanosheets. GO-wrapped FMPSi (FMPSi@GO) was loaded with a cisplatin (Cis) anticancer agent, and Cis-loaded FMPSi@GO (FMPSi-Cis@GO) exhibited the dual stimuli (pH and NIR)-responsiveness of controlled drug release, i.e., the drug release rate was distinctly enhanced at acidic pH 5.5 than at neutral pH 7.0 and further enhanced under NIR irradiation at acidic pH condition. Notably, dequalinium-conjugated FMPSi-Cis@GO (FMPSi-Cis@GO@DQA) demonstrated an excellent specificity for mitochondrial targeting in cancer cells without noticeable toxicity to normal human cells. Our novel silicon nanocarriers demonstrated not only stimuli (pH and NIR)-responsive controlled drug release, but also selective accumulation in the mitochondria of cancer cells and destroying them. Full article
(This article belongs to the Special Issue Sustainable Materials and Technologies for Drug Delivery and Tissue Engineering)
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Figure 1

Figure 1
<p>(<b>A</b>) FT-IR spectra of MPSi, FMPSi, and FMPSi-Cis@GO; (<b>B</b>) Raman spectra of MPSi, FMPSi, and FMPSi-Cis@GO; (<b>C</b>) Nitrogen adsorption/desorption isotherms and pore size distribution using the Barrett–Joyner–Halenda (BJH) analysis of as-prepared samples (MSN, MPSi, and FMPSi); and (<b>D</b>) TEM images: (<b>1</b>) MSN, (<b>2</b>) FMPSi, (<b>3</b>) FMPSi@GO, and (<b>4</b>) FMPSi-Cis@GO@DQA.</p> Full article ">Figure 2
<p>(<b>A</b>) Temperature changes of pure PBS, MSN, MPSi, and FMPSi@GO solutions during NIR light exposure (808 nm) at a power density of 1.0 W/cm<sup>2</sup> for 20 min; (<b>B</b>) Cis release profiles of FMPSi-Cis@GO in PBS at pH 7.4 and pH 5.5; (<b>C</b>) Cumulative release fractions of FMPSi-Cis@GO in PBS at pH 5.5 and pH 7.4 before and after NIR irradiation (808 nm laser, 1.0 W/cm<sup>2</sup>) for 20 min. The dotted lines of purple color indicate the temperature changes of in vitro solution by the periodic irradiations of NIR light for 20 min; (<b>D</b>) Model fits of release profiles of FMPSi-Cis at pH 5.5 and pH 7.4 and release profiles of FMPSi-Cis@GO in PBS (pH 5.5) at 37 °C; and Schematic representation of stimuli-responsive controlled drug release from FMPSi-Cis@GO; (<b>E</b>) pH effects on drug release mechanism of FMPSi-based NPs; and (<b>F</b>) NIR irradiation effect on drug release mechanism of FMPSi-based NPs.</p> Full article ">Figure 3
<p>Cellular uptake and cytotoxicity studies: (<b>A</b>) Representative confocal microscopy image of HeLa cells after treating with FMPSi-Cis@GO (<b>left</b>) and FMPSi-Cis@GO@DQA (<b>right</b>) at 4 h, 8 h, and 12 h incubation; (<b>B</b>) Cell viability profile of HeLa, SH-S5Y5, and HEK293 cells treated with different concentrations of as-prepared NPs for 48 h. Data are represented as the mean ± SD (<span class="html-italic">n</span> = 3).</p> Full article ">Figure 4
<p>The rationale for mitochondria-targeting of multifunctional mesoporous silicon nanoparticle system to assess Δψm. (<b>A</b>) FITC fluorescence absorption intensities of isolated mitochondria measured from HeLa, SH-SY5Y, and HEK293 cells without and with the indicated treatments; (<b>B</b>) Mitochondrial membrane potential of HeLa and SH-SY5Y cells after treatment with FMPSi-Cis@GO and FMPSi-Cis@GO@DQA for a short time of 24 h incubation. PBS treatment was the negative (control) group; FCCP treatment was a positive control. Δψm was measured by fluorescence intensity after cells were stained with TMRE. Data are represented as the mean ± SD (<span class="html-italic">n</span> = 3); and (<b>C</b>) Schematic illustration of specific mitochondrion targeting and therapeutic effects of multifunctional mesoporous silicon nanoparticle system.</p> Full article ">Figure 5
<p>Cross-section of TEM images of multifunctional silicon NPs in the endosome and mitochondria in HeLa cells, with the magnified TEM images of red square boxes (<b>a</b>,<b>b</b>) Control MPSi; (<b>c</b>,<b>d</b>) FMPSi-Cis@GO; and (<b>e</b>,<b>f</b>) FMPSi-Cis@GO@DQA. HeLa cells were treated with 50 μg/mL NPs. CM and CN represent cell membrane and cell nucleus, respectively. Red and yellow arrows indicate NPs, and blue arrows indicate mitochondria.</p> Full article ">Scheme 1
<p>Schematic illustration of the consecutive steps for the synthesis of multifunctional MPSi NPs (FMPSi, FMPSi@GO, and FMPSi@GO@DQA) and targeted drug delivery to mitochondria of the cancer cell. The coating mechanism of GO and the therapeutic effects of DQA are also presented.</p> Full article ">
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