Pharmaceutics

33 pages, 3853 KiB

Open AccessReview

Functionalization of Nanoparticulate Drug Delivery Systems and Its Influence in Cancer Therapy

by Theodora Amanda Seidu, Perpetua Takunda Kutoka, Dorothy Owusu Asante, Muhammad Asim Farooq, Raphael N. Alolga and Wang Bo

Pharmaceutics 2022, 14(5), 1113; https://doi.org/10.3390/pharmaceutics14051113 - 23 May 2022

Cited by 28 | Viewed by 4799

Abstract

Research into the application of nanocarriers in the delivery of cancer-fighting drugs has been a promising research area for decades. On the other hand, their cytotoxic effects on cells, low uptake efficiency, and therapeutic resistance have limited their therapeutic use. However, the urgency [...] Read more.

Research into the application of nanocarriers in the delivery of cancer-fighting drugs has been a promising research area for decades. On the other hand, their cytotoxic effects on cells, low uptake efficiency, and therapeutic resistance have limited their therapeutic use. However, the urgency of pressing healthcare needs has resulted in the functionalization of nanoparticles’ (NPs) physicochemical properties to improve clinical outcomes of new, old, and repurposed drugs. This article reviews recent research on methods for targeting functionalized nanoparticles to the tumor microenvironment (TME). Additionally, the use of relevant engineering techniques for surface functionalization of nanocarriers (liposomes, dendrimers, and mesoporous silica) and their critical roles in overcoming the current limitations in cancer therapy—targeting ligands used for targeted delivery, stimuli strategies, and multifunctional nanoparticles—were all reviewed. The limitations and future perspectives of functionalized nanoparticles were also finally discussed. Using relevant keywords, published scientific literature from all credible sources was retrieved. A quick search of the literature yielded almost 400 publications. The subject matter of this review was addressed adequately using an inclusion/exclusion criterion. The content of this review provides a reasonable basis for further studies to fully exploit the potential of these nanoparticles in cancer therapy. Full article

► Show Figures

Figure 1

Figure 1
Graphical representation of the abstract. Illustration of tumor microenvironment and how surface-functionalized nanocarriers containing antitumor drugs actively target tumor cells. Full article ">Figure 2
Nanoparticles’ classifications, applications, and differences. Full article ">Figure 3
Targeted liposomes having two peptides (TfR- and VEGFR2-specified peptides) and two antitumor agents (doxorubicin and vincristine). (a) Cellular uptake (After 2 h at 37 °C, cellular uptake of Cy5.5-loaded liposomes of varying densities of T7 (A) and DA7R (B) in C6 cells. The cells’ auto-fluorescence was used as the control. Cellular uptake of varied Cy5.5 loaded lyposomes by bEND.3 cells (C), HUVECs (D), and C6 (E) cells); (b) cytotoxicity study (The cytotoxic activity of free DOX + free VCR, as well as some liposomes containing DOX and VCR); (c) biodistribution study (The biodistribution of Cy5.5 in varied liposomes in mice with intracranial C6 glioma was ascertained using an IVIS® Spectrum-CT (A). A CLSM was used to show the allocation of Cy5.5 in the brains of mice with intracranial C6 glioma (B). 16 days after inoculation, MRI of physiologic and pathological brains (C). Glioma tumor cell division rate in the brain (D). Survival curves according to Kaplan–Meier (E). The yellow line represents the intracranial glioma margin, and the arrow represents the glioma cells. The red is Cy5.5, and the nuclei are stained with DAPI (blue). Effectiveness after treatment with different formulations at 1 mg/kg (DOX 0.8 mg/kg + VCR 0.2 mg/kg) on days 8, 10, 12, and 14 after inoculation.) This designed system could go through the blood–brain barrier and blood–tumor barrier, with enhanced cellular uptake and cytotoxicity [<a href="#B32-pharmaceutics-14-01113" class="html-bibr">32</a>]. Copyright 2017, Taylor & Francis Journals. * p < 0.05. Full article ">Figure 4
Illustration of the types of functionalization on the nanoparticulate drug delivery systems. Full article ">

23 pages, 995 KiB

Open AccessReview

Heterogeneity of In Vitro Expanded Mesenchymal Stromal Cells and Strategies to Improve Their Therapeutic Actions

by Laura Olmedo-Moreno, Yolanda Aguilera, Carmen Baliña-Sánchez, Alejandro Martín-Montalvo and Vivian Capilla-González

Pharmaceutics 2022, 14(5), 1112; https://doi.org/10.3390/pharmaceutics14051112 - 23 May 2022

Cited by 29 | Viewed by 3778

Abstract

Beneficial properties of mesenchymal stromal cells (MSCs) have prompted their use in preclinical and clinical research. Accumulating evidence has been provided for the therapeutic effects of MSCs in several pathologies, including neurodegenerative diseases, myocardial infarction, skin problems, liver disorders and cancer, among others. [...] Read more.

Beneficial properties of mesenchymal stromal cells (MSCs) have prompted their use in preclinical and clinical research. Accumulating evidence has been provided for the therapeutic effects of MSCs in several pathologies, including neurodegenerative diseases, myocardial infarction, skin problems, liver disorders and cancer, among others. Although MSCs are found in multiple tissues, the number of MSCs is low, making in vitro expansion a required step before MSC application. However, culture-expanded MSCs exhibit notable differences in terms of cell morphology, physiology and function, which decisively contribute to MSC heterogeneity. The changes induced in MSCs during in vitro expansion may account for the variability in the results obtained in different MSC-based therapy studies, including those using MSCs as living drug delivery systems. This review dissects the different changes that occur in culture-expanded MSCs and how these modifications alter their therapeutic properties after transplantation. Furthermore, we discuss the current strategies developed to improve the beneficial effects of MSCs for successful clinical implementation, as well as potential therapeutic alternatives. Full article

(This article belongs to the Special Issue Living Cell-Based Drug Delivery Systems for Biomedical Applications)

► Show Figures

Figure 1

10 pages, 4857 KiB

Open AccessArticle

Preparation and Drug Release Profile of Chitosan–Siloxane Hybrid Capsules Coated with Hydroxyapatite

by Yuki Shirosaki, Yasuyo Tsukatani, Kohei Okamoto, Satoshi Hayakawa and Akiyoshi Osaka

Pharmaceutics 2022, 14(5), 1111; https://doi.org/10.3390/pharmaceutics14051111 - 23 May 2022

Cited by 2 | Viewed by 2053

Abstract

Chitosan is a cationic polymer that forms polymerized membranes upon reaction with anionic polymers. Chitosan−carboxymethyl cellulose (CMC) capsules are drug delivery carrier candidates whose mechanical strength and permeability must be controlled to achieve sustained release. In this study, the capsules were prepared from [...] Read more.

Chitosan is a cationic polymer that forms polymerized membranes upon reaction with anionic polymers. Chitosan−carboxymethyl cellulose (CMC) capsules are drug delivery carrier candidates whose mechanical strength and permeability must be controlled to achieve sustained release. In this study, the capsules were prepared from chitosan−γ-glycidoxypropyltrimethoxysilane (GPTMS)−CMC. The mechanical stability of the capsules was improved by crosslinking the chitosan with GPTMS. The capsules were then coated with hydroxyapatite (HAp) by alternately soaking them in calcium chloride solution and disodium hydrogen phosphate solution to prevent rapid initial drug release. Cytochrome C (CC), as a model drug, was introduced into the capsules via two routes, impregnation and injection, and then the CC released from the capsules was examined. HAp was found to be deposited on the internal and external surfaces of the capsules. The amount of CC introduced, and the release rate were reduced by the HAp coating. The injection method was found to result in the greatest CC loading. Full article

(This article belongs to the Special Issue Innovative Drug Delivery Systems for Regenerative Medicine)

► Show Figures

Figure 1

13 pages, 2569 KiB

Open AccessArticle

High Performance Gold Nanorods@DNA Self-Assembled Drug-Loading System for Cancer Thermo-Chemotherapy in the Second Near-Infrared Optical Window

by Wei Chang, Junfeng Wang, Jing Zhang, Qing Ling, Yumei Li and Jie Wang

Pharmaceutics 2022, 14(5), 1110; https://doi.org/10.3390/pharmaceutics14051110 - 23 May 2022

Cited by 8 | Viewed by 2795

Abstract

In terms of synergistic cancer therapy, biological nanomaterials with a second near-infrared (NIR-II) window response can greatly increase photothermal effects and photoacoustic imaging performance. Herein, we report a novel stimuli-responsive multifunctional drug-loading system which was constructed by integrating miniature gold nanorods (GNR) as [...] Read more.

In terms of synergistic cancer therapy, biological nanomaterials with a second near-infrared (NIR-II) window response can greatly increase photothermal effects and photoacoustic imaging performance. Herein, we report a novel stimuli-responsive multifunctional drug-loading system which was constructed by integrating miniature gold nanorods (GNR) as the NIR-II photothermal nanorods and cyclic ternary aptamer (CTA) composition as a carrier for chemotherapy drugs. In this system, doxorubicin hydrochloride (DOX, a chemotherapy drug) binds to the G-C base pairs of the CTA, which exhibited a controlled release behavior based on the instability of G-C base pairs in the slightly acidic tumor microenvironment. Upon the 1064 nm (NIR-II biowindow) laser irradiation, the strong photothermal and promoted cargo release properties endow gold nanorods@CTA (GNR@CTA) nanoparticles displaying excellent synergistic anti-cancer effect. Moreover, the GNR@CTA of NIR also possesses thermal imaging and photoacoustic (PA) imaging properties due to the strong NIR region absorbance. This work enables to obtaining a stimuli-responsive “all-in-one” nanocarrier, which are promising candidate for bimodal imaging diagnosis and chemo-photothermal synergistic therapy. Full article

(This article belongs to the Special Issue Polymer and Lipid-based Materials for Nanodrug Delivery Systems)

► Show Figures

Figure 1

Figure 1
Construction and characterization of GNR@CTA. (a) Schematic diagram of CTA, (b) Functional area division of CTA, (c) Gel Electrophoresis Characterization of CTA, (d) TEM characterization of GNR, (e) Length and width distribution of GNR, and (f) UV-visible-NIR spectrum of GNR. Full article ">Figure 2
Loading and release of DOX by GNR@CTA. (a) Fluorescence analysis of GNP@CTA drug loading and NIR photothermal release. (b) Analysis of drug loading efficiency of GNR@CTA. (c) Analysis of GNR@CTA drug loading stability. Red “*” indicates free DOX. Full article ">Figure 3
Intracellular drug delivery and in vivo imaging of GNR@CTA. (a) CLSM analysis of GNR@CTA(DOX) specific targeted drug delivery. (b) Infrared thermal imaging of GNR@CTA in vivo. (c) PAI imaging analysis of GNR@CTA in vivo. Full article ">Figure 4
Analysis of anti-tumor therapeutic effect of different materials in nude mice model. (a) Nude mice’s weight change curve. (b) Changes in tumor size in different groups. (c) Comparison of nude mice before and after the experiment, the tumor is marked by the red circle. (d) Comparison of tumor tissue sections before and after treatment. (e) Compare the tissue sections of the main organs between the experimental group and healthy nude mice. Full article ">Scheme 1
Schematic illustration of the fabrication process and GNR@CTA synergistic anticancer therapy. Full article ">

26 pages, 3170 KiB

Open AccessReview

Bioinspired and Biomimetic Nanomedicines for Targeted Cancer Therapy

by Xiaoqiu Xu, Tong Li and Ke Jin

Pharmaceutics 2022, 14(5), 1109; https://doi.org/10.3390/pharmaceutics14051109 - 23 May 2022

Cited by 24 | Viewed by 3829

Abstract

Undesirable side effects and multidrug resistance are the major obstacles in conventional chemotherapy towards cancers. Nanomedicines provide alternative strategies for tumor-targeted therapy due to their inherent properties, such as nanoscale size and tunable surface features. However, the applications of nanomedicines are hampered in [...] Read more.

Undesirable side effects and multidrug resistance are the major obstacles in conventional chemotherapy towards cancers. Nanomedicines provide alternative strategies for tumor-targeted therapy due to their inherent properties, such as nanoscale size and tunable surface features. However, the applications of nanomedicines are hampered in vivo due to intrinsic disadvantages, such as poor abilities to cross biological barriers and unexpected off-target effects. Fortunately, biomimetic nanomedicines are emerging as promising therapeutics to maximize anti-tumor efficacy with minimal adverse effects due to their good biocompatibility and high accumulation abilities. These bioengineered agents incorporate both the physicochemical properties of diverse functional materials and the advantages of biological materials to achieve desired purposes, such as prolonged circulation time, specific targeting of tumor cells, and immune modulation. Among biological materials, mammalian cells (such as red blood cells, macrophages, monocytes, and neutrophils) and pathogens (such as viruses, bacteria, and fungi) are the functional components most often used to confer synthetic nanoparticles with the complex functionalities necessary for effective nano-biointeractions. In this review, we focus on recent advances in the development of bioinspired and biomimetic nanomedicines (such as mammalian cell-based drug delivery systems and pathogen-based nanoparticles) for targeted cancer therapy. We also discuss the biological influences and limitations of synthetic materials on the therapeutic effects and targeted efficacies of various nanomedicines. Full article

(This article belongs to the Special Issue Frontier Novelties of Nanotechnology in Cancer Targeting)

► Show Figures

Figure 1

19 pages, 7666 KiB

Open AccessArticle

Reparative Efficacy of Liposome-Encapsulated Oleanolic Acid against Liver Inflammation Induced by Fine Ambient Particulate Matter and Alcohol in Mice

by Ching-Ting Wei, Yu-Wen Wang, Yu-Chiuan Wu, Li-Wei Lin, Chia-Chi Chen, Chun-Yin Chen and Shyh-Ming Kuo

Pharmaceutics 2022, 14(5), 1108; https://doi.org/10.3390/pharmaceutics14051108 - 23 May 2022

Cited by 8 | Viewed by 3106

Abstract

Airborne fine particulate matter (PM_2.5) is a severe problem and is associated with health issues including liver diseases. Workers performing manual labor tend to be alcohol consumers during work, where they are also exposed to PM_2.5. Long-term PM_2.5 [...] Read more.

Airborne fine particulate matter (PM_2.5) is a severe problem and is associated with health issues including liver diseases. Workers performing manual labor tend to be alcohol consumers during work, where they are also exposed to PM_2.5. Long-term PM_2.5 exposure can increase oxidative stress, leading to inflammation. Whether long-term exposure to air pollution and alcohol synergistically increases liver fibrosis risk warrants investigation. Oleanolic acid (OA)—a triterpenoid—has antioxidant and anti-inflammatory activities, but its low water solubility and cytotoxicity impair its potential applications. In this study, we fabricated liposomal OA nanoparticles (Lipo-OAs); then, we evaluated the anti-inflammatory effect on exposed cells and the ameliorative effect of Lipo-OAs on PM_2.5 and alcohol-induced liver fibrosis in mice. The half maximal inhibitory concentration of PM_2.5 for hepatic stellate cells was 900 μg/mL; at a concentration of ≥600 μg/mL, PM_2.5 significantly increased interleukin-6 and tumor necrosis factor-α production. OA encapsulation in Lipo-OAs, 353 ± 140 nm in diameter with 79% encapsulation efficiency, significantly reduced OA cytotoxicity. Lipo-OAs treatment significantly reduced alanine aminotransferase, aspartate aminotransferase, and γ-glutamyltransferase levels; histologically, it alleviated steatosis and improved Ishak’s modified HAI score. In conclusion, Lipo-OAs have potential anti-inflammatory and reparative effects for PM_2.5 and alcohol-induced liver injury treatment. Full article

(This article belongs to the Special Issue Nanoformulation of Drug Delivery Systems for Natural Products)

► Show Figures

Figure 1

15 pages, 5375 KiB

Open AccessArticle

Simultaneous Improvement of Dissolution Behavior and Oral Bioavailability of Antifungal Miconazole via Cocrystal and Salt Formation

by Ksenia V. Drozd, Alex N. Manin, Denis E. Boycov and German L. Perlovich

Pharmaceutics 2022, 14(5), 1107; https://doi.org/10.3390/pharmaceutics14051107 - 22 May 2022

Cited by 22 | Viewed by 3356

Abstract

Miconazole shows low oral bioavailability in humans due to poor aqueous solubility, although it has demonstrated various pharmacological activities such as antifungal, anti-tubercular and anti-tumor effects. Cocrystal/salt formation is one of the effective methods for solving this problem. In this study, different methods [...] Read more.

Miconazole shows low oral bioavailability in humans due to poor aqueous solubility, although it has demonstrated various pharmacological activities such as antifungal, anti-tubercular and anti-tumor effects. Cocrystal/salt formation is one of the effective methods for solving this problem. In this study, different methods (liquid-assisted grinding, slurrying and lyophilization) were used to investigate their impact on the formation of the miconazole multicomponent crystals with succinic, maleic and dl-tartaric acids. The solid state of the prepared powder was characterized by differential scanning calorimetry, powder X-ray diffraction and scanning electron microscopy. It was found that lyophilization not only promotes partial amorphization of both salts but also allows obtaining a new polymorph of the miconazole salt with dl-tartaric acid. The lyophilized salts compared with the same samples prepared by two other methods showed better dissolution rates but low stability during the studies due to rapid recrystallization. Overall, it was determined that the preparation method of multicomponent crystals affects the solid-state characteristics and miconazole physicochemical properties significantly. The in vivo studies revealed that the miconazole multicomponent crystals indicated the higher peak blood concentration and area under the curve from 0 to 32 h values 2.4-, 2.9- and 4.6-fold higher than the pure drug. Therefore, this study demonstrated that multicomponent crystals are promising formulations for enhancing the oral bioavailability of poorly soluble compounds. Full article

(This article belongs to the Special Issue Applications of Crystal Engineering in Drug Delivery)

► Show Figures

Graphical abstract

Graphical abstract
Full article ">Figure 1
Chemical structures of miconazole and dicarboxylic acids used in this study. Full article ">Figure 2
Comparison of the freeze-dried powders of (a) [MCL + SucAc] cocrystal (2:1), (b) [MCL + MlcAc] salt (1:1) and (c) [MCL + TartAc] salt (1:1) obtained from TBA/H2O mixtures with different TBA content. Full article ">Figure 3
DSC curves of the MCL multicomponent crystals: (a) [MCL + SucAc] cocrystal (2:1), (b) [MCL + MlcAc] salt (1:1), (c) [MCL + TartAc] salt (1:1) prepared via LAG (black line), slurrying (red line) and freeze-drying (blue line). Full article ">Figure 4
SEM images of the MCL multicomponent crystals: (a) [MCL + SucAc] cocrystal (2:1), (b) [MCL + MlcAc] salt (1:1) and (c) [MCL + TartAc] salt (1:1), prepared by LAG, slurring and freeze-drying (from left to right). Full article ">Figure 5
PXRD patterns for the MCL salts: (a) [MCL + MlcAc] (1:1) and (b) [MCL + TartAc] (1:1) in crystalline forms (black line) and freeze-dried samples (red line) studied as a function of storage time. Grey stripes highlight some unique peaks different from the peaks of the crystalline [MCL + TartAc] salt. Full article ">Figure 6
Time profiles of the MCL dissolved concentrations from its multicomponent crystals prepared via different methods in aqueous solution pH 6.8 at 37 °C: (a) [MCL + SucAc] (2:1), (b) [MCL + MlcAc] (1:1), (c) [MCL + TartAc] (1:1). Full article ">Figure 7
Mean plasma concentration-time profiles of the MCL and its multicomponent crystals after oral administration to rabbits. Key: –●–—pure MCL, –▼–—[MCL + SucAc] (2:1), –■–—[MCL + MlcAc] (1:1), –♦–—[MCL + TartAc] (1:1). Full article ">

12 pages, 1681 KiB

Open AccessArticle

Evaluation of 3-Borono-l-Phenylalanine as a Water-Soluble Boron Neutron Capture Therapy Agent

by Naoya Kondo, Fuko Hirano and Takashi Temma

Pharmaceutics 2022, 14(5), 1106; https://doi.org/10.3390/pharmaceutics14051106 - 22 May 2022

Cited by 19 | Viewed by 4653

Abstract

Although 4-borono-l-phenylalanine (4-BPA) is currently the only marketed agent available for boron neutron capture therapy (BNCT), its low water solubility raises concerns. In this study, we synthesized 3-borono-l-phenylalanine (3-BPA), a positional isomer of 4-BPA, with improved water solubility. We [...] Read more.

Although 4-borono-l-phenylalanine (4-BPA) is currently the only marketed agent available for boron neutron capture therapy (BNCT), its low water solubility raises concerns. In this study, we synthesized 3-borono-l-phenylalanine (3-BPA), a positional isomer of 4-BPA, with improved water solubility. We further evaluated its physicochemical properties, tumor accumulation, and biodistribution. The water solubility of 3-BPA was 125 g/L, which is more than 100 times higher than that of 4-BPA. Due to the high water solubility, we prepared the administration solution of 3-BPA without a solubilizer sugar, which is inevitably added to 4-BPA preparation and has adverse effects. In in vitro and in vivo experiments, boron accumulation in cancers after administration was statistically equivalent in both sugar-complexed 3-BPA and 4-BPA. Furthermore, the biodistribution of 3-BPA was comparable with that of sugar-complexed 3-BPA. Since 3-BPA has high water solubility and tumor targetability equivalent to 4-BPA, 3-BPA can replace 4-BPA in future BNCT. Full article

(This article belongs to the Special Issue Novel Anticancer Strategies (Volume II))

► Show Figures

Figure 1

Figure 1
Chemical structure of 4-borono-l-phenylalanine (4-BPA, (a)) and 3-borono-l-phenylalanine (3-BPA, (b)). Full article ">Figure 2
In vitro cell uptake of 3-BPA and 4-BPA in cancer cells with various LAT1-expression. (a) The LAT1/β-actin ratio of each cell calculated from the expression levels of LAT1 and β-actin obtained by Western blotting and standardized to a value of 1 for T3M-4. (b,c) LAT1-specific accumulation (the difference in accumulation between the inhibitor group and the noninhibitor group) in T3M-4, A549, B16F10, and U-87MG cells after 1, 5, and 30 min incubation with 3-BPA (b) and 4-BPA (c). (d) The relationship between the uptake (% dose/mg protein) of 3-BPA and 4-BPA at 30 min incubation with a simple linear regression. Statistical analyses of LAT1-specific accumulation of 3-BPA (b) and 4-BPA (c) were shown in <a href="#app1-pharmaceutics-14-01106" class="html-app">Supplementary Tables S3 and S4</a>, respectively. Full article ">Figure 3
Boron accumulation in B16F10 melanoma (a) and plasma (b), and melanoma to plasma ratio of boron accumulation (c) of 3-BPA-Fru and 4-BPA-Fru at 10, 30, 60, and 120 min after administration. Full article ">Figure 4
Boron accumulation in T3M-4 tumor, plasma, liver, kidneys, and the tissues surrounding the tumor at 60 min after administration of 3-BPA, 3-BPA-Fru, and 4-BPA-Fru into BALB/c nu-nu mice. * p < 0.001 by Tukey’s multiple comparison tests. Full article ">

10 pages, 674 KiB

Open AccessArticle

Impact of Magnesium on Oxytocin Receptor Function

by Vimala N. Bharadwaj, Justin Meyerowitz, Bende Zou, Michael Klukinov, Ni Yan, Kaustubh Sharma, David J. Clark, Xinmin Xie and David C. Yeomans

Pharmaceutics 2022, 14(5), 1105; https://doi.org/10.3390/pharmaceutics14051105 - 21 May 2022

Cited by 9 | Viewed by 3705

Abstract

Background and Purpose: The intranasal administration of oxytocin (OT) reduces migraine headaches through activation of the oxytocin receptor (OTR). Magnesium ion (Mg²⁺⁾ concentration is critical to the activation of the OTR, and a low serum Mg²⁺ concentration is predictive of a [...] Read more.

Background and Purpose: The intranasal administration of oxytocin (OT) reduces migraine headaches through activation of the oxytocin receptor (OTR). Magnesium ion (Mg²⁺⁾ concentration is critical to the activation of the OTR, and a low serum Mg²⁺ concentration is predictive of a migraine headache. We, therefore, examined the functional impact of Mg²⁺ concentration on OT-OTR binding efficacy using two complimentary bioassays. Experimental Approach: Current clamp recordings of rat trigeminal ganglia (TG) neurons measured the impact of Mg²⁺ on an OT-induced reduction in excitability. In addition, we assessed the impact of Mg²⁺ on intranasal OT-induced craniofacial analgesia in rats. Key Results: While OT alone dose-dependently hyperpolarized TG neurons, decreasing their excitability, the addition of 1.75 mM Mg²⁺ significantly enhanced this effect. Similarly, while the intranasal application of OT produced dose-dependent craniofacial analgesia, Mg²⁺ significantly enhanced these effects. Conclusions and Implications: OT efficacy may be limited by low ambient Mg²⁺ levels. The addition of Mg²⁺ to OT formulations may improve its efficacy in reducing headache pain as well as for other OT-dependent processes. Full article

(This article belongs to the Special Issue Intranasal Administration: Potential Route as Diverse Targeting Systems)

► Show Figures

Graphical abstract

Graphical abstract
Full article ">Figure 1
Effect of addition of Mg2+ to the OT-induced decrease in excitability of TG neurons from CFA-inflamed rats. (A,B) Examples of current clamp traces of TG neurons from CFA-inflamed rat; (A) no change in membrane potential when treated with 3 nM OT in a 0 mM MgCl2 buffer and (B) hyperpolarization observed when treated with 3 nM OT in a 1.75 mM MgCl2 buffer. (C,D) OT alone dose-dependently hyperpolarizes TG cell membranes, decreasing excitability; the addition of 1.75 mM Mg2+ significantly (* p < 0.05, ANOVA) potentiates the capacity of OT to hyperpolarize TG cell membranes (C). A specific example of this is observed in (D), where the addition of Mg2+ significantly (* p < 0.05) increased the induced membrane hyperpolarization of 1 nM or 3 nM OT from −1.4 ± 1.6 mV (n = 6) and −2.5 ± 0.6 mV (n = 6) for OT alone to −8.1 ± 1.2 mV (n = 7) and −9.3 ± 1.2 mV (n = 7) for OT plus Mg2+, respectively. Subsequent pairwise comparisons indicated significant (* p < 0.05) differences from OT alone for OT plus Mg2+ at 1, 3, 10, and 1000 nM OT concentrations. This enhanced hyperpolarization is emblematic of decreased excitability and thus, decreased capacity to carry pain signals to the central nervous system for pain perception. Error bars show ± S.D. Full article ">Figure 2
Effect of the addition of Mg2+ on intranasal OT-induced craniofacial analgesia. For both A-delta (A) and C fiber (B) mediated withdrawal responses to noxious heat stimulation of the cheek of pre-inflamed rats, intranasally applied OT produced a dose-dependent analgesic effect as evidenced by significant (p < 0.05, ANOVA) increases in withdrawal latency at the 60 min time point after administration, n = 10. However, the addition of 300 mM Mg2+ to the treatment significantly increased OT analgesia for both stimulus types (p < 0.05). Subsequent pairwise comparisons indicated significant (* = p < 0.05) differences from OT alone for OT plus Mg2+ for A-delta testing at OT doses of 0.5, 1.0, and 32 μg (p < 0.05); C-fiber responses were significantly different (* = p < 0.05) at 0.5, 4.0, 8.0, and 32 μg OT. Interestingly, while the efficacy of the highest OT dose (32 μg) demonstrated a decrease in efficacy when compared to lower doses, this dose-response inversion was prevented by the addition of Mg2+. Full article ">

19 pages, 5211 KiB

Open AccessArticle

Green Synthesis of Silver Nanoparticles Using Hypericum perforatum L. Aqueous Extract with the Evaluation of Its Antibacterial Activity against Clinical and Food Pathogens

by Abdalrahim Alahmad, Wael A. Al-Zereini, Tahani J. Hijazin, Osama Y. Al-Madanat, Ibrahim Alghoraibi, Omar Al-Qaralleh, Samer Al-Qaraleh, Armin Feldhoff, Johanna-Gabriela Walter and Thomas Scheper

Pharmaceutics 2022, 14(5), 1104; https://doi.org/10.3390/pharmaceutics14051104 - 21 May 2022

Cited by 37 | Viewed by 5069

Abstract

The rapid development of nanotechnology and its applications in medicine has provided the perfect solution against a wide range of different microbes, especially antibiotic-resistant ones. In this study, a one-step approach was used in preparing silver nanoparticles (AgNPs) by mixing silver nitrate with [...] Read more.

The rapid development of nanotechnology and its applications in medicine has provided the perfect solution against a wide range of different microbes, especially antibiotic-resistant ones. In this study, a one-step approach was used in preparing silver nanoparticles (AgNPs) by mixing silver nitrate with hot Hypericum perforatum (St. John’s wort) aqueous extract under high stirring to prevent agglomeration. The formation of silver nanoparticles was monitored by continuous measurement of the surface plasma resonance spectra (UV-VIS). The effect of St. John’s wort aqueous extract on the formation of silver nanoparticles was evaluated and fully characterized by using different physicochemical techniques. The obtained silver nanoparticles were spherical, monodisperse, face-centered cubic (fcc) crystal structures, and the size ranges between 20 to 40 nm. They were covered with a capping layer of organic compounds considered as a nano dimension protective layer that prevents agglomeration and sedimentation. AgNPs revealed antibacterial activity against both tested Gram-positive and Gram-negative bacterial strains causing the formation of 13–32 mm inhibition zones with MIC 6.25–12.5 µg/mL; Escherichia coli strains were resistant to tested AgNPs. The specific growth rate of S. aureus was significantly reduced due to tested AgNPs at concentrations ≥½ MIC. AgNPs did not affect wound migration in fibroblast cell lines compared to control. Our results highlighted the potential use of AgNPs capped with plant extracts in the pharmaceutical and food industries to control bacterial pathogens’ growth; however, further studies are required to confirm their wound healing capability and their health impact must be critically evaluated. Full article

(This article belongs to the Special Issue Pharmaceutical Applications of Plant Extracts)

► Show Figures

Graphical abstract

18 pages, 3616 KiB

Open AccessArticle

Surface Functionalization of Silica Nanoparticles: Strategies to Optimize the Immune-Activating Profile of Carrier Platforms

by Benjamin Punz, Litty Johnson, Mark Geppert, Hieu-Hoa Dang, Jutta Horejs-Hoeck, Albert Duschl and Martin Himly

Pharmaceutics 2022, 14(5), 1103; https://doi.org/10.3390/pharmaceutics14051103 - 21 May 2022

Cited by 8 | Viewed by 3469

Abstract

Silica nanoparticles (SiNPs) are generally regarded as safe and may represent an attractive carrier platform for nanomedical applications when loaded with biopharmaceuticals. Surface functionalization by different chemistries may help to optimize protein loading and may further impact uptake into the targeted tissues or [...] Read more.

Silica nanoparticles (SiNPs) are generally regarded as safe and may represent an attractive carrier platform for nanomedical applications when loaded with biopharmaceuticals. Surface functionalization by different chemistries may help to optimize protein loading and may further impact uptake into the targeted tissues or cells, however, it may also alter the immunologic profile of the carrier system. In order to circumvent side effects, novel carrier candidates need to be tested thoroughly, early in their development stage within the pharmaceutical innovation pipeline, for their potential to activate or modify the immune response. Previous studies have identified surface functionalization by different chemistries as providing a plethora of modifications for optimizing efficacy of biopharmaceutical (nano)carrier platforms while maintaining an acceptable safety profile. In this study, we synthesized SiNPs and chemically functionalized them to obtain different surface characteristics to allow their application as a carrier system for allergen-specific immunotherapy. In the present study, crude natural allergen extracts are used in combination with alum instead of well-defined active pharmaceutical ingredients (APIs), such as recombinant allergen, loaded onto (nano)carrier systems with immunologically inert and stable properties in suspension. This study was motivated by the hypothesis that comparing different charge states could allow tailoring of the binding capacity of the particulate carrier system, and hence the optimization of biopharmaceutical uptake while maintaining an acceptable safety profile, which was investigated by determining the maturation of human antigen-presenting cells (APCs). The functionalized nanoparticles were characterized for primary and hydrodynamic size, polydispersity index, zeta potential, endotoxin contamination. As potential candidates for allergen-specific immunotherapy, the differently functionalized SiNPs were non-covalently coupled with a highly purified, endotoxin-free recombinant preparation of the major birch pollen allergen Bet v 1 that functioned for further immunological testing. Binding efficiencies of allergen to SiNPs was controlled to determine uptake of API. For efficacy and safety assessment, we employed human monocyte-derived dendritic cells as model for APCs to detect possible differences in the particles’ APC maturation potential. Functionalization of SiNP did not affect the viability of APCs, however, the amount of API physisorbed onto the nanocarrier system, which induced enhanced uptake, mainly by macropinocytosis. We found slight differences in the maturation state of APCs for the differently functionalized SiNP–API conjugates qualifying surface functionalization as an effective instrument for optimizing the immune response towards SiNPs. This study further suggests that surface-functionalized SiNPs could be a suitable, immunologically inert vehicle for the efficient delivery of biopharmaceutical products, as evidenced here for allergen-specific immunotherapy. Full article

(This article belongs to the Special Issue Application of Nanomedicine in Immunotherapy: Recent Advances and Prospects)

► Show Figures

Graphical abstract

Graphical abstract
Full article ">Figure 1
TEM images of the differently functionalized SiNPs for determination of primary particle size (A) SiNP, (B) SiNP_A, (C) SiNP_M, Size bar: 100 nm. Full article ">Figure 2
Quantification of LPS content by (A) HEK blue assay and (B) monocyte activation test (MAT) to analyze LPS contamination in the SiNP samples and Bet v 1. The LPS content was quantified based on LPS standard curves. The data are presented as mean ± SD (n = 4). Full article ">Figure 3
The binding efficiencies of differently functionalized SiNP samples were determined (A) directly from the pellet by SDS-PAGE and (B) indirectly from the supernatant by BCA assay. The data are presented as mean ± SD (n = 4) * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001. Full article ">Figure 4
Viability of the moDCs tested with (A) LDH assay based on the release of LDH and (B) Live/dead staining via flow cytometry after stimulation of moDCs with the samples after 24 h. The negative control in the experiment was dead cells (incubated at 95 °C for 10 min) and the positive control (viable cells) was unstimulated moDCs. Full article ">Figure 5
Impact on the uptake of allergen by moDCs. (A) Kinetics of uptake represented as mean fluorescence intensity (MFI) over the course of 24 h with SiNP-Bet, SiNP_M-Bet, and Bet. (B) Inhibition of uptake with the endocytosis inhibitors chlorpromazine hydrochloride (CPZ), cytochalasin D (CytoD), filipin, and rottlerin after stimulation with moDCs for 24 h (n = 4 individual donors). The data are presented as mean ± SD (* p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001). Full article ">Figure 6
Co-stimulatory molecule expression by stimulated moDCs with functionalized SiNPs and Bet. (A) CD40, (B) CD86, (C) CD83, (D) HLA-DR, and (E) CD80 were analyzed. LPS was used as the positive control and a statistical significance was observed in comparison to the cell control indicating a successful stimulation. All particles and conjugates were compared to Bet v 1 for statistical analysis (n = 6 individual donors) (* p ≤ 0.05; ** p ≤ 0.01; **** p ≤ 0.0001). Full article ">Figure 7
Cyto-/chemokine release of APCs. (A) IL8, (B) MCP-1, (C) MIP-1α, (D) MIP-1 β, (E) IL-4, (F) IL-6, (G) TNFα, (H) IL1-RA. LPS was used as the positive control and a statistical significance was observed in comparison to the cell control, indicating successful stimulation. The particles were compared to Bet v 1 for statistical analysis (n = 5 individual donors) (* p ≤ 0.05, *** p ≤ 0.001; **** p ≤ 0.0001). Full article ">

22 pages, 4743 KiB

Open AccessEditor’s ChoiceArticle

Ultrasound Triggers Hypericin Activation Leading to Multifaceted Anticancer Activity

by Federica Foglietta, Roberto Canaparo, Simone Cossari, Patrizia Panzanelli, Franco Dosio and Loredana Serpe

Pharmaceutics 2022, 14(5), 1102; https://doi.org/10.3390/pharmaceutics14051102 - 21 May 2022

Cited by 16 | Viewed by 2662

Abstract

The use of ultrasound (US) in combination with a responsive chemical agent (sonosensitizer) can selectively trigger the agent’s anticancer activity in a process called sonodynamic therapy (SDT). SDT shares some properties with photodynamic therapy (PDT), which has been clinically approved, but sets itself [...] Read more.

The use of ultrasound (US) in combination with a responsive chemical agent (sonosensitizer) can selectively trigger the agent’s anticancer activity in a process called sonodynamic therapy (SDT). SDT shares some properties with photodynamic therapy (PDT), which has been clinically approved, but sets itself apart because of its use of US rather than light to achieve better tissue penetration. SDT provides anticancer effects mainly via the sonosensitizer-mediated generation of reactive oxygen species (ROS), although the precise nature of the underpinning mechanism is still under debate. This work investigates the SDT anticancer activity of hypericin (Hyp) in vitro in two- (2D) and three-dimensional (3D) HT-29 colon cancer models, and uses PDT as a yardstick due to its well-known Hyp phototoxicity. The cancer cell uptake and cellular localization of Hyp were investigated first to determine the proper noncytotoxic concentration and incubation time of Hyp for SDT. Furthermore, ROS production, cell proliferation, and cell death were evaluated after Hyp was exposed to US. Since cancer relapse and transporter-mediated multidrug resistance (MDR) are important causes of cancer treatment failure, the US-mediated ability of Hyp to elicit immunogenic cell death (ICD) and overcome MDR was also investigated. SDT showed strong ROS-mediated anticancer activity 48 h after treatment in both the HT-29 models. Specific damage-associated molecular patterns that are consistent with ICD, such as calreticulin (CRT) exposure and high-mobility group box 1 protein (HMGB1) release, were observed after SDT with Hyp. Moreover, the expression of the ABC transporter, P-glycoprotein (P-gp), in HT-29/MDR cells was not able to hinder cancer cell responsiveness to SDT with Hyp. This work reveals, for the first time, the US responsiveness of Hyp with significant anticancer activity being displayed, making it a full-fledged sonosensitizer for the SDT of cancer. Full article

(This article belongs to the Special Issue Novel Strategies to Improve the Therapeutic Effects of Natural Anticancer Compounds)

► Show Figures

Graphical abstract

12 pages, 2971 KiB

Open AccessArticle

Development of an LNP-Encapsulated mRNA-RBD Vaccine against SARS-CoV-2 and Its Variants

by Cong Liu, Nino Rcheulishvili, Zhigao Shen, Dimitri Papukashvili, Fengfei Xie, Ziqian Wang, Xingyun Wang, Yunjiao He and Peng George Wang

Pharmaceutics 2022, 14(5), 1101; https://doi.org/10.3390/pharmaceutics14051101 - 20 May 2022

Cited by 17 | Viewed by 4722

Abstract

Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is undoubtedly the most challenging pandemic in the current century and remains a global health emergency. As the number of COVID-19 cases in the world is on the rise and [...] Read more.

Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is undoubtedly the most challenging pandemic in the current century and remains a global health emergency. As the number of COVID-19 cases in the world is on the rise and variants continue to emerge, there is an urgent need for vaccines. Among all immunization approaches, mRNA vaccines have demonstrated more promising results in response to this challenge. Herein, we designed an mRNA-based vaccine encoding the receptor-binding domain (RBD) of SARS-CoV-2 encapsulated in lipid nanoparticles (LNPs). Intramuscular (i.m.) administration of the mRNA-RBD vaccine elicited broad-spectrum neutralizing antibodies and cellular responses against not only the wild-type SARS-CoV-2 virus but also Delta and Omicron variants. These results indicated that two doses of mRNA-RBD immunization conferred a strong immune response in mice against the wild-type SARS-CoV-2, while the booster dose provided a sufficient immunity against SARS-CoV-2 and its variants. Taken together, the three-dose regimen strategy of the mRNA-RBD vaccine proposed in the present study appears to be a promising reference for the development of mRNA vaccines targeting SARS-CoV-2 variants. Full article

(This article belongs to the Special Issue Sustainable Materials and Technologies for Drug Delivery and Tissue Engineering)

► Show Figures

Figure 1

Figure 1
Preparation and characterization of SARS-CoV-2 mRNA-RBD vaccine. (a) The construct of mRNA-RBD vaccine for COVID-19. (b) The expression of RBD mRNA in HEK293T cells (c) The size distribution of LNPs was measured by a Malvern particle size instrument. (d) The encapsulation efficiency of LNPs using the ribogreen assay (e) The zeta potential for LNPs at pH 7.4 PBS. (f) Cryo-TEM images of the LNPs (scale bar, 100 nm, 50 nm). (g) Time-dependent change in particle size of LNPs upon storage at 4 °C, −20 °C and −80 °C. (h) Time-dependent change in encapsulation efficiency of LNPs for 14 days using the ribogreen assay upon storage at 4 °C, −20 °C and −80 °C. UTR, untranslated region; NC, negative control. Full article ">Figure 2
In vivo delivery of mRNA-LNPs formulation. (a) Duration and expression pattern of LNPs-encapsulated Fluc mRNA in mice injected by i.v. and i.m. routes. BALB/c mice were injected with 10 µg of FLuc mRNA encapsulated by LNPs by the intramuscular (i.m.) and intravenous (i.v.) routes and representative IVIS images. (b) Representative Fluc expression in different organs for FLuc mRNA-LNP injected mice under IVIS imaging. Full article ">Figure 3
m1Ψ substitution of U improves mRNA expression. (a) Chemical structures of the U, C, and 2 modified nucleobase (m1Ψ, m5C). (b) Fluorescence microscope analyses of modified EGFP mRNAs harboring U, m5C, and m1Ψ translated in HEK293T cells. (c) IVIS analyses of modified FLuc mRNAs harboring U, m5C, and m1Ψ translated in HEK293T cells. m1Ψ, N1-methylpseudouridine; U, uridine; C, cytidine; EGFP, enhanced green fluorescent protein; FLuc, firefly luciferase. Full article ">Figure 4
Humoral immune response in mRNA-RBD vaccinated mice. (a) Mice immunization and sample collection schedule (b) SARS-CoV-2 RBD-specific IgG concentrations were assayed by ELISA. (c) Inhibition of RBD-hACE2 interaction by sera from mRNA-RBD vaccinated mice. (d) Pseudovirus neutralization assay of the mRNA-RBD group shows the EC50 titers for the SARS-CoV-2, Delta, and Omicron pseudovirus. NAb, neutralizing antibodies; IgG, immunoglobulin G. (** p < 0.01; *** p < 0.001; **** p < 0.0001, n.s. no significance.) Full article ">Figure 5
Cell immune response in mice by mRNA-RBD vaccines. (a) The proportions of CD3+CD4+ and CD3+CD8+ T cells were tested by flow cytometry. (b) The percentages of IFNγ-producing CD4+ and CD8+ T cells were detected by intracellular cytokine staining. LNP, lipid nanoparticle; RBD, receptor-binding domain. (** p < 0.01; *** p < 0.001). Full article ">

27 pages, 3770 KiB

Open AccessArticle

Anti-Cancerous Potential of Polysaccharides Derived from Wheat Cell Culture

by Alima Murtazina, Gloria Ruiz Alcala, Yaiza Jimenez-Martinez, Juan Antonio Marchal, Anel Tarabayeva, Elmira Bitanova, Gordon McDougall, Nazira Bishimbayeva and Houria Boulaiz

Pharmaceutics 2022, 14(5), 1100; https://doi.org/10.3390/pharmaceutics14051100 - 20 May 2022

Cited by 7 | Viewed by 2992

Abstract

There is a global need to discover effective anti-cancerous compounds from natural sources. Cultivated wheat cells can be a valuable source of non-toxic or low toxic plant-derived polysaccharides. In this study, we evaluated the anti-cancer ability of seven fractions of wheat cell culture [...] Read more.

There is a global need to discover effective anti-cancerous compounds from natural sources. Cultivated wheat cells can be a valuable source of non-toxic or low toxic plant-derived polysaccharides. In this study, we evaluated the anti-cancer ability of seven fractions of wheat cell culture polysaccharides (WCCPSs) in the HCT-116 colon cancer cell line. Almost all (6/7) fractions had an inhibitory effect on the proliferation of colon cancer cells, and two fractions (A-b and A-f) had considerable therapeutic indexes. The WCCPS fractions induced cell cycle arrest in the G1 phase and induced different rates of apoptosis (≤48%). Transmission and scanning electron microscopy revealed that WCCPS fractions caused apoptotic changes in the nucleus and cytoplasm, including damage to mitochondria and external morphological signs of apoptosis. In addition, the WCCPSs induced an increase in the levels of Bax, cytochrome c, and caspases 8 and 3, indicating that cell death progressed through intrinsic and extrinsic pathways of apoptosis. Furthermore, some fractions caused a significant decrease of c-Myc, b-catenin, NFkB2, and HCAM (CD 44) levels, indicating enhanced cell differentiation. Thus, for the first time, our results provide a proof of concept of the anti-cancer capacity of WCCPS fractions in colorectal cancer. Full article

(This article belongs to the Special Issue Novel Strategies to Improve the Therapeutic Effects of Natural Anticancer Compounds)

► Show Figures

Figure 1

Figure 1
Scheme of PS fractions obtainment. Full article ">Figure 2
Inhibition of HCT-116 colon cancer cell line and CCD118 CO normal colon cell line proliferation (in %) under the influence of different concentrations of T−010, B−010, and UB−010 fractions. Fractionated PSs (T−010—total fraction, B−010—bound fraction, UB−010—unbound fraction). (*** p < 0.001, ** p < 0.01 and * p < 0.05 versus control). Full article ">Figure 3
Inhibitory activity of PS fractions on the normal colon cell line CCD118 CO, compared with inhibition of colon cancer cells of the HCT−116 line. T−010 fraction (A), UB−fraction (B), T−b fraction (C), A−b fraction (D), T−f fraction (E); A−f fraction (F); (A,C,E) T−fractions; (D,F) A−fractions; UB–unbound fraction (**** p < 0.0001, *** p < 0.001, ** p < 0.01 and * p < 0.05 versus control). Full article ">Figure 4
Cell cycle and apoptosis of HCT-116 colon cancer cells treated by WCCPS. (A)—untreated cells, (B)—A−b fraction, (C)–T−010 fraction, (D)—UB−010 fraction, (E)—T−b fraction (2× IC50), (F)—A−f fraction, (G)—T−f fraction, (H)—cell cycle, (I)—debris, (J)—apoptosis. (*** p < 0.001, ** p < 0.01 and * p < 0.05 versus control). Full article ">Figure 4 Cont.
Cell cycle and apoptosis of HCT-116 colon cancer cells treated by WCCPS. (A)—untreated cells, (B)—A−b fraction, (C)–T−010 fraction, (D)—UB−010 fraction, (E)—T−b fraction (2× IC50), (F)—A−f fraction, (G)—T−f fraction, (H)—cell cycle, (I)—debris, (J)—apoptosis. (*** p < 0.001, ** p < 0.01 and * p < 0.05 versus control). Full article ">Figure 5
Transmission electron microscopy of the HCT-116 cell line treated by WCCPS in concentrations of IC50. (Scale bar: (a,b,e,j,k,l–r)—400, (c,d,f)—600, (g–i)—200, (s–w)—5000 nm), (a)—nucleus of untreated cells, (b,c)—nucleus with condensed chromatin and nuclear pores, apoptotic blebs of cells treated by T-010, (d)—nucleus of cells treated by T-b fraction, condensation chromatin, nuclear pores (Np) (2× IC50), (e)—nucleus of cells treated by A-b fraction, (f)—blebs in intercellular space between cells treated by UB-010 fraction, (g)—mitochondria of non-treated cells, (h)—mitochondria treated by T-010, (i)—mitochondria treated by T-b fraction, (j)—Golgi apparatus of non-treated cell, (k)—swollen Golgi apparatus in cells treated by T-010, (l) –Goldi (A-b fraction), (m)—Golgi apparatus (T-b fraction), (n)—lysosomes and autophagic vacuole of untreated cell, (o)—accumulation of multiple autophagic vacuoles with double and single membrane (T-010), (p)—autophagic vacuoles with double and triple membrane, (q)—vesicles in cell treated by T-b fraction, (r)—double membrane autophagic large vesicle in cell treated by UB-010 fraciton, (s)—untreated cell, (t)—cells treated by A-f fraction—brush borders, (u)—tight junctions between cells (A-f fraction), (v)—cryptae structures (A-f fraction), (w)—cells treated by T-f fraction—plenty of vesicles in intercellular space; (A)—the diameter of cross section of mitochondria, (B)—number of damaged mitochondria per picture. N-nucleus, Np—nuclear pores, M—mitochondria, G—Golgi apparatus, L—lysosome, av—autophagic vesicles; ALV—autophagic large vesicle, arrow—tight cell junctions. (** p < 0.01 and * p < 0.05 versus control). Full article ">Figure 6
Scanning electron microscopy of HCT-116 cells treated by PS fractions. Magnification ×3000, 50 µm: (a)—untreated cells, (b)—T-010 fraction, (c)—A-b fraction, (d)—UB-010 fraction, (e)—T-b fraction IC50, (f)—T-b fraction with 2× IC50; Magnification ×9975, 10 µm: (g)—untreated cell, (h)—T-010 fraction, (i)—A-b fraction, (j)—UB-D fraction, (k)—T-b in concentration of IC50, (l)—T-b fraction 2 IC50, (A)—number of microvilli per 10 µm. (*** p < 0.001, ** p < 0.01 and * p < 0.05 versus control). Full article ">Figure 7
Western blot of different proteins related to cancer cell proliferation after treatment with PS fractions. Western blot quantification was normalized against the β-actin signal. Data were obtained from three independent experiments performed in duplicate and are expressed as mean ± SD from three independent experiments performed in triplicate (**** p < 0.0001, *** p < 0.001, ** p < 0.01 and * p < 0.05 versus control). (a) membrane that contain proteins of NFkB p100, c-Myc, Cyt C; (b) membrane with CD-44, Caspases 3 and 8, and Bax, (c) membrane with beta-catenin. Full article ">

28 pages, 4445 KiB

Open AccessArticle

Crystallization of Form II Paracetamol with the Assistance of Carboxylic Acids toward Batch and Continuous Processes

by Kuan-Lin Yeh, Hung-Lin Lee and Tu Lee

Pharmaceutics 2022, 14(5), 1099; https://doi.org/10.3390/pharmaceutics14051099 - 20 May 2022

Cited by 4 | Viewed by 2936

Abstract

Form II paracetamol has captured the interest of researchers due to its improved compressibility. However, its low stability has made it difficult to be produced on a large scale with good reproducibility. In the present study, the selective polymorphic formation of paracetamol was [...] Read more.

Form II paracetamol has captured the interest of researchers due to its improved compressibility. However, its low stability has made it difficult to be produced on a large scale with good reproducibility. In the present study, the selective polymorphic formation of paracetamol was carried out by cooling crystallization with four types of additives: adipic acid, fumaric acid, oxalic acid, and succinic acid. It was found that: (1) the more additives that were added, the higher the probability of forming Form II paracetamol; (2) Form II paracetamol could be induced by seeding the paracetamol aqueous solution with Form II paracetamol and fumaric acid crystals, and not the other three carboxylic acids; (3) a new solution complex of paracetamol–oxalic acid, evidenced by the solubility diagram, was responsible for the selective nucleation of Form II paracetamol in the oxalic acid aqueous solution; and (4) the range of the degree of supersaturation for nucleating Form II paracetamol was extended with the assistance of oxalic acid or fumaric acid. In large-scale crystallization, Form II paracetamol was produced by the continuous crystallization of 44 mg of paracetamol/mL in 50 wt% of fumaric acid aqueous solution with a flow rate of 150 mL/min. Full article

(This article belongs to the Special Issue Advances in Pharmaceutical Solid Forms)

► Show Figures

Graphical abstract

19 pages, 3268 KiB

Open AccessArticle

Design, Synthesis, and Evaluation of Linker-Optimised PSMA-Targeting Radioligands

by Fanny Lundmark, Gustav Olanders, Sara Sophie Rinne, Ayman Abouzayed, Anna Orlova and Ulrika Rosenström

Pharmaceutics 2022, 14(5), 1098; https://doi.org/10.3390/pharmaceutics14051098 - 20 May 2022

Cited by 9 | Viewed by 3353

Abstract

Prostate-specific membrane antigen (PSMA) is overexpressed in the majority of prostate cancer cells and is considered to be an important target for the molecular imaging and therapy of prostate cancer. Herein, we present the design, synthesis, and evaluation of 11 PSMA-binding radioligands with [...] Read more.

Prostate-specific membrane antigen (PSMA) is overexpressed in the majority of prostate cancer cells and is considered to be an important target for the molecular imaging and therapy of prostate cancer. Herein, we present the design, synthesis, and evaluation of 11 PSMA-binding radioligands with modified linker structures, focusing on the relationship between molecular structure and targeting properties. The linker design was based on 2-naphthyl-L-alanine-tranexamic acid, the linker structure of PSMA-617. X-ray crystal-structure analysis of PSMA and structure-based design were used to generate the linker modifications, suggesting that substitution of tranexamic acid could lead to interactions with Phe546, Trp541, and Arg43 within the binding cavity. After synthesis through SPPS, analogues were labelled with indium-111 and evaluated in vitro for their specific binding, affinity, and cellular retention. Selected compounds were further evaluated in vivo in PSMA-expressing tumour-bearing mice. Based on the results, 2-naphthyl-L-alanine appears to be crucial for good targeting properties, whereas tranexamic acid could be replaced by other substituents. [¹¹¹In]In-BQ7859, consisting of a 2-naphthyl-L-alanine-L-tyrosine linker, demonstrated favourable targeting properties. The substitution of tranexamic acid for L-tyrosine in the linker led to an improved tumour-to-blood ratio, highlighting [¹¹¹In]In-BQ7859 as a promising PSMA-targeting radioligand. Full article

(This article belongs to the Section Drug Targeting and Design)

► Show Figures

Figure 1

44 pages, 10378 KiB

Open AccessReview

Recent Advancements in Microneedle Technology for Multifaceted Biomedical Applications

by Deepak Kulkarni, Fouad Damiri, Satish Rojekar, Mehrukh Zehravi, Sarker Ramproshad, Dipali Dhoke, Shubham Musale, Ashiya A. Mulani, Pranav Modak, Roshani Paradhi, Jyotsna Vitore, Md. Habibur Rahman, Mohammed Berrada, Prabhanjan S. Giram and Simona Cavalu

Pharmaceutics 2022, 14(5), 1097; https://doi.org/10.3390/pharmaceutics14051097 - 20 May 2022

Cited by 60 | Viewed by 10249

Abstract

Microneedle (MNs) technology is a recent advancement in biomedical science across the globe. The current limitations of drug delivery, like poor absorption, low bioavailability, inadequate skin permeation, and poor biodistribution, can be overcome by MN-based drug delivery. Nanotechnology made significant changes in fabrication [...] Read more.

Microneedle (MNs) technology is a recent advancement in biomedical science across the globe. The current limitations of drug delivery, like poor absorption, low bioavailability, inadequate skin permeation, and poor biodistribution, can be overcome by MN-based drug delivery. Nanotechnology made significant changes in fabrication techniques for microneedles (MNs) and design shifted from conventional to novel, using various types of natural and synthetic materials and their combinations. Nowadays, MNs technology has gained popularity worldwide in biomedical research and drug delivery technology due to its multifaceted and broad-spectrum applications. This review broadly discusses MN’s types, fabrication methods, composition, characterization, applications, recent advancements, and global intellectual scenarios. Full article

(This article belongs to the Special Issue Recent Advances in Microneedle-Mediated Drug Delivery)

► Show Figures

Figure 1

17 pages, 5933 KiB

Open AccessArticle

Topography-Mediated Enhancement of Nonviral Gene Delivery in Stem Cells

by Lu Ge, Liangliang Yang, Reinier Bron and Patrick van Rijn

Pharmaceutics 2022, 14(5), 1096; https://doi.org/10.3390/pharmaceutics14051096 - 20 May 2022

Cited by 3 | Viewed by 2573

Abstract

Gene delivery holds great promise for bioengineering, biomedical applications, biosensors, diagnoses, and gene therapy. In particular, the influence of topography on gene delivery is considered to be an attractive approach due to low toxicity and localized delivery properties. Even though many gene vectors [...] Read more.

Gene delivery holds great promise for bioengineering, biomedical applications, biosensors, diagnoses, and gene therapy. In particular, the influence of topography on gene delivery is considered to be an attractive approach due to low toxicity and localized delivery properties. Even though many gene vectors and transfection systems have been developed to enhance transfection potential and combining it with other forms of stimulations could even further enhance it. Topography is an interesting surface property that has been shown to stimulate differentiation, migration, cell morphology, and cell mechanics. Therefore, it is envisioned that topography might also be able to stimulate transfection. In this study, we tested the hypothesis “topography is able to regulate transfection efficiency”, for which we used nano- and microwave-like topographical substrates with wavelengths ranging from 500 nm to 25 µm and assessed the transfectability of human bone marrow-derived mesenchymal stem cells (hBM-MSCs) and myoblasts. For transfection, Lipofectamine 2000 and a gene encoding plasmid for red-fluorescent protein (m-Cherry) were used and topography-induced cell morphology and transfection efficiency was analyzed. As a result, topography directs cell spreading, elongation, and proliferation as well as the transfection efficiency, which were investigated but were found not to be correlated and dependent on the cell type. A 55% percent improvement of transfection efficiency was identified for hBM-MSCs grown on 2 µm wrinkles (24.3%) as compared to hBM-MSCs cultured on flat controls (15.7%). For myoblast cells, the highest gene-expression efficiency (46.1%) was observed on the 10 µm topography, which enhanced the transfection efficiency by 64% as compared to the flat control (28.1%). From a qualitative assessment, it was observed that the uptake capacity of cationic complexes of TAMRA-labeled oligodeoxynucleotides (ODNs) was not topography-dependent but that the intracellular release was faster, as indicated by the positively stained nuclei on 2 μm for hBM-MSCs and 10 μm for myoblasts. The presented results indicate that topography enhances the gene-delivery capacity and that the responses are dependent on cell type. This study demonstrates the important role of topography on cell stimulation for gene delivery as well as understanding the uptake capacity of lipoplexes and may be useful for developing advanced nonviral gene delivery strategies. Full article

(This article belongs to the Collection Drug Delivery in The Netherlands)

► Show Figures

Figure 1

Figure 1
(A) AFM images and amplitude curves of the structured PDMS surfaces obtained after imprinting. (B) Wavelength and (C) Amplitude of created wave-like surface. Data are reported as mean ± standard deviation (SD) (n = 30 wrinkles). Scale bar is 10 μm and applies to all images. Full article ">Figure 2
Influence of nano- and micropatterns on the morphology of hBM-MSCs and myoblast cells. (A) Representative fluorescence microscopy images of hBM-MSCs and myoblast cells grown on different wrinkle surfaces. F-actin and cell nucleus were stained with TRITC-labeled phalloidin (red) and by DAPI (blue), respectively. (B) Cell area of hBM-MSCs and (C) myoblast cells grown on different topographies. (D) Cell aspect ratio (CAR) of hBM-MSCs and (E) Myoblast cells grown on different topographies. Data are shown as mean ± standard deviation (SD), and * p < 0.05, ** p < 0.01, *** p < 0.001. Scale bar is 100 μm for all images. Full article ">Figure 3
Influence of nano- and micropatterns on the transfection capacity of hBM-MSCs. (A) Representative fluorescence microscopy images of transfect hBM-MSCs after 24 h. F-actin and cell nucleus were stained with FITC-labeled phalloidin (green) and by DAPI (blue), respectively. The transfected cells are m-cheery express cells (red). (B) Flow cytometry profiles obtained after the treatment with LF2000/pDNA encoding m-cheery proteins. The percentage of transfected cells was evaluated and hBM-MSCs cells without a transfection agent were selected as the control. (C) Quantification data of transfected cells grown on different topographies. Three independent experiments were presented. Data are shown as mean ± standard deviation (SD), and * p < 0.05, ** p < 0.01, *** p < 0.001. Scale bar is 100 μm and applies to all images. Full article ">Figure 4
Influence of nano- and micropatterns on the transfection capacity of myoblast cells. (A) Representative fluorescent images of transfected myoblast cells on wave-like PDMS. F-actin and cell nuclei were stained with FITC-labeled phalloidin (green) and by DAPI (blue), respectively. Successfully transfected cells are expressed as m-Cherry (red). (B) Flow cytometry profiles obtained after the treatment with LF2000/p-DNA encoding m-Cherry proteins. (C) Quantification data of transfected cells grown on different topographies. Three independent experiments were per-formed. Data are shown as mean ± standard deviation (SD), and * p < 0.05, ** p < 0.01, *** p < 0.001. Scale bar is 100 μm and applies to all images. Full article ">Figure 5
Influence of nano- and micropatterns on the cellular uptake capacity of cationic complexes. (A) Representative fluorescence images of hBM-MSCs cells cultured on wave-like PDMS, showing the cellular uptake of oligonucleotides (red). F-actin was stained with FITC-labeled phalloidin (green). (B) Representative fluorescence images of myoblast cells cultured on wave-like PDMS showing cellular uptake of oligonucleotides (red). F-actin was stained with FITC-labeled phalloidin (green). Scale bar is 100 μm and applies to all images. White arrows mark positive nuclei. Full article ">Figure 6
Cell proliferation assay. (A) Representative fluorescence images hBM-MSCs cells cultured on W0.5, W2, and W25 for 12 h, 24 h, and 48 h. The ki67 positive cells were stained in red, and cell nuclei were counterstained in blue (DAPI). (B) Quantification results of the percentage of Ki67 positive cells of hBM-MSCs at 12 h, 24 h, and 48 h for the chosen substrate. (C) Representative fluorescence images myoblast cells cultured on W0.5, W10, and W25 for 12 h, 24 h, and 48 h. (D) Quantification results of the percentage of Ki67 positive cells of myoblast cells at 12 h, 24 h, and 48 h for the chosen substrate. Three independent experiments were performed. Data are shown as mean ± standard deviation (SD), and * p < 0.05, ** p < 0.01, *** p < 0.001, n.s. no significant difference. Scale bar is 100 μm and applies to all images. Full article ">

24 pages, 2467 KiB

Open AccessEditor’s ChoiceReview

Controlled-Release Nanosystems with a Dual Function of Targeted Therapy and Radiotherapy in Colorectal Cancer

by Pedro Cruz-Nova, Alejandra Ancira-Cortez, Guillermina Ferro-Flores, Blanca Ocampo-García and Brenda Gibbens-Bandala

Pharmaceutics 2022, 14(5), 1095; https://doi.org/10.3390/pharmaceutics14051095 - 20 May 2022

Cited by 13 | Viewed by 3751

Abstract

Nanoparticles are excellent platforms for several biomedical applications, including cancer treatment. They can incorporate different molecules to produce combinations of chemotherapeutic agents, radionuclides, and targeting molecules to improve the therapeutic strategies against cancer. These specific nanosystems are designed to have minimal side effects [...] Read more.

Nanoparticles are excellent platforms for several biomedical applications, including cancer treatment. They can incorporate different molecules to produce combinations of chemotherapeutic agents, radionuclides, and targeting molecules to improve the therapeutic strategies against cancer. These specific nanosystems are designed to have minimal side effects on healthy cells and better treatment efficacy against cancer cells when compared to chemotherapeutics, external irradiation, or targeted radiotherapy alone. In colorectal cancer, some metal and polymeric nanoparticle platforms have been used to potentialize external radiation therapy and targeted drug delivery. Polymeric nanoparticles, liposomes, albumin-based nanoparticles, etc., conjugated with PEG and/or HLA, can be excellent platforms to increase blood circulation time and decrease side effects, in addition to the combination of chemo/radiotherapy, which increases therapeutic efficacy. Additionally, radiolabeled nanoparticles have been conjugated to target specific tissues and are mainly used as agents for diagnosis, drug/gene delivery systems, or plasmonic photothermal therapy enhancers. This review aims to analyze how nanosystems are shaping combinatorial therapy and evaluate their status in the treatment of colorectal cancer. Full article

(This article belongs to the Special Issue Novel Strategies for Cancer Targeted Delivery)

► Show Figures

Figure 1

Figure 1
Physical and chemical interactions of nanoparticles for functionalization, drug loading, or radiolabeling. Physical methods include adsorption, interaction charge, and hydrophobic or hydrophilic interactions. Chemical bonds can be formed by carbodiimide coupling. Full article ">Figure 2
Mechanism of gold nanoparticles functionalization by covalent bonding. The sodium citrate acts as a stabilizer when it is absorbed into AuNPs. Subsequently, a second chemical reaction with the sulfhydryl group of functionalization molecules reacts with metallic gold forming the Au–S covalent bond. Full article ">Figure 3
AuNP functionalization with amphiphilic molecules. Amphiphilic molecules (bilayer or small micelles) are absorbed into the AuNp surface and can act as stabilizers or ligands. Full article ">Figure 4
Nanoparticle systems for combined chemoradiotherapy in colorectal cancer. The chemotherapeutic agent can be loaded or conjugated to the nanoparticle and can be carried to the cancer cell through ligands, antibodies, and/or molecular recognition agents that interact with overexpressed proteins in colorectal cancer tumors. The combination with radiation therapy can be through external X-rays or by using radiolabeled molecules. Full article ">Figure 5
Main components of targeted radiolabeled nanoparticles. Radionuclides can be conjugated directly onto the nanoparticle surface, with or without a spacer. Full article ">

18 pages, 5196 KiB

Open AccessArticle

Comparison of Paliperidone Palmitate from Different Crystallization Processes and Effect on Formulations In Vitro and In Vivo

by Junfeng Shi, Dan Wang, Yang Tian, Zengming Wang, Jing Gao, Nan Liu, Xiang Gao, Aiping Zheng, Hui Zhang and Meixian Xiang

Pharmaceutics 2022, 14(5), 1094; https://doi.org/10.3390/pharmaceutics14051094 - 20 May 2022

Cited by 5 | Viewed by 3858

Abstract

The quality of active pharmaceutical ingredients (APIs) is an important factor which can affect the safety and efficacy of pharmaceuticals. This study was designed to investigate the nature of paliperidone palmitate (PP) obtained by different crystallization processes, then compare the characteristics between test [...] Read more.

The quality of active pharmaceutical ingredients (APIs) is an important factor which can affect the safety and efficacy of pharmaceuticals. This study was designed to investigate the nature of paliperidone palmitate (PP) obtained by different crystallization processes, then compare the characteristics between test formulations which prepared PP of different crystallization and reference formulations (Invega Sustenna^®) in vitro and in vivo. Two different PPs, namely PP-1 and PP-2, were prepared by different crystallization methods. Contact angle, morphology, and crystallinity of the PPs were characterized. Taking the particle sizes and distribution of Invega Sustenna^® as reference, test formulations were prepared by the wet milling method using either a PP-1 or PP-2 sample. Their release behavior, stability in vitro, and pharmacokinetics in vivo were subsequently investigated. The results indicated that PP-2 had a higher surface free energy (SFE). More small particles were attached to the PP-1 surface under the influence of crystallization temperature. Different crystallization processes did not change the crystal of PP, but changed the crystallinity of PP. There was no obvious difference in in vitro releases between test formulations. However, the stability and state of formulation containing PP-2 were better compared to formulations containing PP-1, indicated by differences in crystallinity and SFE. Meanwhile, pharmacokinetic in vivo results demonstrated that the pharmacokinetic profiles and parameters of formulation containing PP-2 and Invega Sustenna^® tended to be consistent, but those of formulations containing PP-1 were significantly different from those of formulations containing PP-2 or Invega Sustenna^®, and there was burst release phenomenon of formulations containing PP-1 in rats. PP made by different crystallization processes could induce changes in appearance, SFE, and crystallinity, and further affect the stability, state, and pharmacokinetic in vivo formulation. Full article

(This article belongs to the Special Issue Advances in Pharmaceutical Solid Forms)

► Show Figures

Figure 1

13 pages, 1113 KiB

Open AccessArticle

Preclinical Assessment of Nebulized Surfactant Delivered through Neonatal High Flow Nasal Cannula Respiratory Support

by Francesca Ricci, Arianna Mersanne, Matteo Storti, Marcello Nutini, Giulia Pellicelli, Angelo Carini, Ilaria Milesi, Marta Lombardini, Raffaele L. Dellacà, Merran A. Thomson, Xabier Murgia, Anna Lavizzari, Federico Bianco and Fabrizio Salomone

Pharmaceutics 2022, 14(5), 1093; https://doi.org/10.3390/pharmaceutics14051093 - 20 May 2022

Cited by 2 | Viewed by 2657

Abstract

High-flow nasal cannula (HFNC) is a non-invasive respiratory support (NRS) modality to treat premature infants with respiratory distress syndrome (RDS). The delivery of nebulized surfactant during NRS would represent a truly non-invasive method of surfactant administration and could reduce NRS failure rates. However, [...] Read more.

High-flow nasal cannula (HFNC) is a non-invasive respiratory support (NRS) modality to treat premature infants with respiratory distress syndrome (RDS). The delivery of nebulized surfactant during NRS would represent a truly non-invasive method of surfactant administration and could reduce NRS failure rates. However, the delivery efficiency of nebulized surfactant during HFNC has not been evaluated in vitro or in animal models of respiratory distress. We, therefore, performed first a benchmark study to compare the surfactant lung dose delivered by commercially available neonatal nasal cannulas (NCs) and HFNC circuits commonly used in neonatal intensive care units. Then, the pulmonary effect of nebulized surfactant delivered via HFNC was investigated in spontaneously breathing rabbits with induced respiratory distress. The benchmark study revealed the surfactant lung dose to be relatively low for both types of NCs tested (Westmed NCs 0.5 ± 0.45%; Fisher & Paykel NCs 1.8 ± 1.9% of a nominal dose of 200 mg/kg of Poractant alfa). The modest lung doses achieved in the benchmark study are compatible with the lack of the effect of nebulized surfactant in vivo (400 mg/kg), where arterial oxygenation and lung mechanics did not improve and were significantly worse than the intratracheal instillation of surfactant. The results from the present study indicate a relatively low lung surfactant dose and negligible effect on pulmonary function in terms of arterial oxygenation and lung mechanics. This negligible effect can, for the greater part, be explained by the high impaction of aerosol particles in the ventilation circuit and upper airways due to the high air flows used during HFNC. Full article

(This article belongs to the Special Issue Advances in Pulmonary Drug Delivery, Volume II)

► Show Figures

Figure 1

Figure 1
Scheme of the experimental setup. PrINT, Premature Infant Nose Throat-Model. Full article ">Figure 2
Pharyngeal pressure measurements during humidified high-flow nasal cannula (HFNC) respiratory support at 8 L/min with different types of commercially available nasal cannulas (NCs) in spontaneously breathing adult rabbits with induced respiratory distress. The dashed line indicates the reference pharyngeal pressure achieved at a nasal continuous positive airway pressure (CPAP) level of 5 cmH2O. No signal of pharyngeal pressure was detected with the Vapothern premature NCs. Three independent pharyngeal pressure measurements were performed for each NC type. Full article ">Figure 3
(A) Mean partial pressure of arterial oxygen (PaO2) and (B) the partial pressure of arterial carbon dioxide (PaCO2) in surfactant-depleted adult rabbits treated with a humidified high-flow nasal cannula (HFNC) only (grey squares), with 200 mg/kg of intratracheal surfactant (black circles), or with 400 mg/kg of nebulized surfactant in combination with HFNC (white triangles). * Between intratracheal surfactant and HFNC-only groups, p < 0.01; # Between intratracheal surfactant and HFNC + nebulized surfactant (400 mg/kg) groups, p < 0.01; § Between HFNC + nebulized surfactant (400 mg/kg) and HFNC-only groups, p < 0.01. Full article ">Figure 4
Box plots showing (A) dynamic compliance (Cdyn) in adult rabbits at baseline, after inducing respiratory distress (after BALs) and 180 min after receiving different treatments, and (B) pressure registered after applying 30 mL of air (PV30 mL) post mortem into the lungs of animals treated with a humidified high-flow nasal cannula (HFNC) only (control, grey boxes), with 200 mg/kg of intratracheal surfactant (SF instillation, black boxes), or with 400 mg/kg of nebulized surfactant in combination with HFNC (white boxes). The boxes display the median (horizontal line) and the first and third quartiles. The x within the boxes indicates the mean of each group, and the whiskers display the maximum and minimum values within the dataset. The dots beyond the whiskers represent outlier values. * Between SF instillation and HFNC-only groups, p < 0.01; # Between SF instillation and HFNC + nebulized surfactant (400 mg/kg) groups, p < 0.01. Full article ">

19 pages, 4617 KiB

Open AccessArticle

Experimental Therapy of HER2-Expressing Xenografts Using the Second-Generation HER2-Targeting Affibody Molecule ¹⁸⁸Re-ZHER2:41071

by Yongsheng Liu, Anzhelika Vorobyeva, Anna Orlova, Mark W. Konijnenberg, Tianqi Xu, Olga Bragina, Annika Loftenius, Erica Rosander, Fredrik Y. Frejd and Vladimir Tolmachev

Pharmaceutics 2022, 14(5), 1092; https://doi.org/10.3390/pharmaceutics14051092 - 20 May 2022

Cited by 9 | Viewed by 3040

Abstract

HER2-targeted radionuclide therapy might be helpful for the treatment of breast, gastric, and ovarian cancers which have developed resistance to antibody and antibody-drug conjugate-based therapies despite preserved high HER2-expression. Affibody molecules are small targeting proteins based on a non-immunoglobulin scaffold. The goal of [...] Read more.

HER2-targeted radionuclide therapy might be helpful for the treatment of breast, gastric, and ovarian cancers which have developed resistance to antibody and antibody-drug conjugate-based therapies despite preserved high HER2-expression. Affibody molecules are small targeting proteins based on a non-immunoglobulin scaffold. The goal of this study was to test in an animal model a hypothesis that the second-generation HER2-targeting Affibody molecule ¹⁸⁸Re-ZHER2:41071 might be useful for treatment of HER2-expressing malignant tumors. ZHER2:41071 was efficiently labeled with a beta-emitting radionuclide rhenium-188 (¹⁸⁸Re). ¹⁸⁸Re-ZHER2:41071 demonstrated preserved specificity and high affinity (K_D = 5 ± 3 pM) of binding to HER2-expressing cells. In vivo studies demonstrated rapid washout of ¹⁸⁸Re from kidneys. The uptake in HER2-expressing SKOV-3 xenografts was HER2-specific and significantly exceeded the renal uptake 4 h after injection and later. The median survival of mice, which were treated by three injections of 16 MBq ¹⁸⁸Re-ZHER2:41071 was 68 days, which was significantly longer (<0.0001 in the log-rank Mantel-Cox test) than survival of mice in the control groups treated with vehicle (29 days) or unlabeled ZHER2:41071 (27.5 days). In conclusion, the experimental radionuclide therapy using ¹⁸⁸Re-ZHER2:41071 enabled enhancement of survival of mice with human tumors without toxicity to the kidneys, which is the critical organ. Full article

(This article belongs to the Special Issue Radiopharmaceuticals for Cancer Imaging and Therapy)

► Show Figures

Graphical abstract

27 pages, 6784 KiB

Open AccessReview

Bioactive Loaded Novel Nano-Formulations for Targeted Drug Delivery and Their Therapeutic Potential

by Sapna Kumari, Anju Goyal, Eda Sönmez Gürer, Evren Algın Yapar, Madhukar Garg, Meenakshi Sood and Rakesh K. Sindhu

Pharmaceutics 2022, 14(5), 1091; https://doi.org/10.3390/pharmaceutics14051091 - 19 May 2022

Cited by 59 | Viewed by 7677

Abstract

Plant-based medicines have received a lot of attention in recent years. Such medicines have been employed to treat medical conditions since ancient times, and in those times only the observed symptoms were used to determine dose accuracy, dose efficacy, and therapy. Rather than [...] Read more.

Plant-based medicines have received a lot of attention in recent years. Such medicines have been employed to treat medical conditions since ancient times, and in those times only the observed symptoms were used to determine dose accuracy, dose efficacy, and therapy. Rather than novel formulations, the current research work on plant-based medicines has mostly concentrated on medicinal active phytoconstituents. In the past recent decades, however, researchers have made significant progress in developing “new drug delivery systems” (NDDS) to enhance therapeutic efficacy and reduce unwanted effects of bioactive compounds. Nanocapsules, polymer micelles, liposomes, nanogels, phytosomes, nano-emulsions, transferosomes, microspheres, ethosomes, injectable hydrogels, polymeric nanoparticles, dendrimers, and other innovative therapeutic formulations have all been created using bioactive compounds and plant extracts. The novel formulations can improve solubility, therapeutic efficacy, bioavailability, stability, tissue distribution, protection from physical and chemical damage, and prolonged and targeted administration, to name a few. The current study summarizes existing research and the development of new formulations, with a focus on herbal bioactive components. Full article

(This article belongs to the Special Issue Nanoformulation of Drug Delivery Systems for Natural Products)

► Show Figures

Figure 1

20 pages, 4882 KiB

Open AccessArticle

Pharmacokinetic/Pharmacodynamic Evaluation of a New Purine-2,6-Dione Derivative in Rodents with Experimental Autoimmune Diseases

by Artur Świerczek, Krzysztof Pociecha, Hanna Plutecka, Marietta Ślusarczyk, Grażyna Chłoń-Rzepa and Elżbieta Wyska

Pharmaceutics 2022, 14(5), 1090; https://doi.org/10.3390/pharmaceutics14051090 - 19 May 2022

Cited by 2 | Viewed by 2647

Abstract

Current treatment strategies of autoimmune diseases (ADs) display a limited efficacy and cause numerous adverse effects. Phosphodiesterase (PDE)4 and PDE7 inhibitors have been studied recently as a potential treatment of a variety of ADs. In this study, a PK/PD disease progression modeling approach [...] Read more.

Current treatment strategies of autoimmune diseases (ADs) display a limited efficacy and cause numerous adverse effects. Phosphodiesterase (PDE)4 and PDE7 inhibitors have been studied recently as a potential treatment of a variety of ADs. In this study, a PK/PD disease progression modeling approach was employed to evaluate effects of a new theophylline derivative, compound 34, being a strong PDE4 and PDE7 inhibitor. Activity of the studied compound against PDE1 and PDE3 in vitro was investigated. Animal models of multiple sclerosis (MS), rheumatoid arthritis (RA), and autoimmune hepatitis were utilized to assess the efficacy of this compound, and its pharmacokinetics was investigated in mice and rats. A new PK/PD disease progression model of compound 34 was developed that satisfactorily predicted the clinical score-time courses in mice with experimental encephalomyelitis that is an animal model of MS. Compound 34 displayed a high efficacy in all three animal models of ADs. Simultaneous inhibition of PDE types located in immune cells may constitute an alternative treatment strategy of ADs. The PK/PD encephalomyelitis and arthritis progression models presented in this study may be used in future preclinical research, and, upon modifications, may enable translation of the results of preclinical investigations into the clinical settings. Full article

(This article belongs to the Section Pharmacokinetics and Pharmacodynamics)

► Show Figures

Figure 1

Figure 1
Chemical structure of compound 34; 4-(1,3-dimethyl-2,6-dioxo-8-(phenethylamino)-2,3,6,7-tetrahydro-1H-purin-7-yl)-N′-(2,3,4-trihydroxybenzylidene)butanehydrazide. Full article ">Figure 2
Schematic presentation of the proposed PK/PD CIA progression model of compound 34 in rats. Full article ">Figure 3
Schematic presentation of the proposed PK/PD MOG35–55-induced EAE progression model of compound 34 in mice. Full article ">Figure 4
PK model fittings to compound 34 serum concentration vs. time profiles. Symbols represent the mean concentrations (±SD) of compound 34 in serum after its IP administration at a single dose of 20 mg·kg−1 in rats (n = 3 per time point) and at a single IP dose of 50 mg·kg−1 in mice (n = 3 mice per time point). Curves depict model fittings. Full article ">Figure 5
The fitting of the PK/PD arthritis progression model to the data and relative body weights of animals from both study groups; (a) time courses of the mean (±SD) relative paw edema in CIA rats (n = 6) after compound 34 treatment at an IP dose of 20 mg·kg−1 once daily for 21 days starting from the 20th day post-immunization, curves depict model fittings, and symbols are measured values; (b,c) are diagnostic plots of the PK/PD arthritis progression model; (d) time courses of the mean (±SD) relative body weight in arthritic rats from experimental and control groups (n = 6). Full article ">Figure 6
The results of the fitting of the PK/PD EAE progression model to the data and relative body weights of mice from the study and control groups; (a) time courses of the mean (+SD) CS in mice with EAE treated with compound 34 at an IP dose of 50 mg·kg−1 once a day for 15 days starting from the 14th day post-immunization. Curves depict model fittings and symbols are measured values; (b,c) are diagnostic plots of the PK/PD EAE progression model; (d) mean (±SD) body weight in mice with EAE (n = 6). Full article ">Figure 7
Mean (+SD) (a) serum TNFα, IFNγ, IL-4, and IL-6 concentrations at 2 h post-ConA administration (n = 4); (b) IFNγ, IL-4, and IL-6 concentrations at 24 h post-ConA administration (n = 4); (c) serum AST and ALT activities at 8 h post-ConA dosing (n = 4). Study group received compound 34 at an IP dose of 50 mg·kg−1 30 min prior to the ConA dosing. * p < 0.05, a Student’s t-test. Full article ">

22 pages, 4189 KiB

Open AccessArticle

The Influence of Short Motifs on the Anticancer Activity of HB43 Peptide

by Claudia Herrera-León, Francisco Ramos-Martín, Hassan El Btaouri, Viviane Antonietti, Pascal Sonnet, Laurent Martiny, Fabrizia Zevolini, Chiara Falciani, Catherine Sarazin and Nicola D’Amelio

Pharmaceutics 2022, 14(5), 1089; https://doi.org/10.3390/pharmaceutics14051089 - 19 May 2022

Cited by 4 | Viewed by 2928

Abstract

Despite the remarkable similarity in amino acid composition, many anticancer peptides (ACPs) display significant differences in terms of activity. This strongly suggests that particular relative dispositions of amino acids (motifs) play a role in the interaction with their biological target, which is often [...] Read more.

Despite the remarkable similarity in amino acid composition, many anticancer peptides (ACPs) display significant differences in terms of activity. This strongly suggests that particular relative dispositions of amino acids (motifs) play a role in the interaction with their biological target, which is often the cell membrane. To better verify this hypothesis, we intentionally modify HB43, an ACP active against a wide variety of cancers. Sequence alignment of related ACPs by ADAPTABLE web server highlighted the conserved motifs that could be at the origin of the activity. In this study, we show that changing the order of amino acids in such motifs results in a significant loss of activity against colon and breast cancer cell lines. On the contrary, amino acid substitution in key motifs may reinforce or weaken the activity, even when the alteration does not perturb the amphipathicity of the helix formed by HB43 on liposomes mimicking their surface. NMR and MD simulations with different membrane models (micelles, bicelles, and vesicles) indicate that the activity reflects the insertion capability in cancer-mimicking serine-exposing membranes, supported by the insertion of N-terminal phenylalanine in the FAK motif and the anchoring to the carboxylate of phosphatidylserine by means of arginine side chains. Full article

(This article belongs to the Special Issue Peptide-Based Drugs for Cancer Therapies)

► Show Figures

Figure 1

Figure 1
HB43-related family of anticancer peptides generated by the ADAPTABLE web server and anticancer activity of its members. (A) Sequence alignment highlighted conserved short motifs that could be at the origin of the activity. (B) ADAPTABLE-generated consensus sequence highlighting the conserved motifs. (C) Sequences of HB43 mutants, designed to alter putative motifs. Alterations are highlighted in red. Full article ">Figure 2
Schematic representation of HB43 and mutants (left) and their respective helical-wheel projections representing alpha-helix structures (right). Hydrophobic amino acids are represented in gray and those with a positive charge (K, R) in blue. Diagrams were created with NetWheels [<a href="#B82-pharmaceutics-14-01089" class="html-bibr">82</a>]. Full article ">Figure 3
Effect of HB43 and mutants on cell viability of (A) SW480, (B) SW620, and (C) HT29 cells. Cells were treated with peptides for 24 h (left) and 48 h (right). Cell viability was measured using the AlamarBlue assay. Results were calculated as percent of control and represented as means ± S.D. of three independent experiments. Full article ">Figure 4
HB43 mutants tend to be unstructured in solution. (A,B,D,E) Chemical shift deviations from random coil values of Hα protons (A,D) and Cα–Cβ carbons (B,E) suggest the absence of the structure of mut3 (top) and mut4 (bottom). (C,F) Schematic representations of mut3 (C) and mut4 (F) are shown as a ‘tube’ colored from blue (N-terminus) to red (C-terminus). Sidechains are shown as sticks with the following color code: positively charged (blue) and nonpolar (light gray). The structures were created with PyMol [<a href="#B76-pharmaceutics-14-01089" class="html-bibr">76</a>]. Data on HB43 peptide were previously published [<a href="#B42-pharmaceutics-14-01089" class="html-bibr">42</a>]. Full article ">Figure 5
mut3 assumes alpha-helical conformation in a lipidic environment. (A) 1H-NMR spectra of mut3 0.8 mM in the presence of DPC at concentrations of 2, 4, 8, 12, 24, and 50 mM. (B) 1H, 1H-NOESY spectrum of mut3 at 278 K in the presence of DPC micelles showing meaningful NOEs in the amide region. (C) MD snapshot of mut3 interacting with DPC micelles. The image was created with PyMol [<a href="#B76-pharmaceutics-14-01089" class="html-bibr">76</a>]. (D) Chemical shift deviations from random coil values of Hα hydrogen atoms. Data on HB43 peptide were previously published [<a href="#B42-pharmaceutics-14-01089" class="html-bibr">42</a>]. Full article ">Figure 6
mut4 assumes alpha-helical conformation in a lipidic environment. (A) 1H-NMR spectra of mut4 0.8 mM in the presence of DPC at concentrations of 2, 4, 8, 12, 24, and 50 mM. (B) 1H, 1H-NOESY spectrum of mut4 at 278 K in the presence of DPC micelles showing meaningful NOEs in the amide region. (C) MD snapshot of mut4 interacting with DPC micelles. The image was created with PyMol [<a href="#B76-pharmaceutics-14-01089" class="html-bibr">76</a>]. (D) Chemical shift deviations from random coil values of Hα hydrogen atoms. Data on HB43 peptide were previously published [<a href="#B42-pharmaceutics-14-01089" class="html-bibr">42</a>]. Full article ">Figure 7
MD snapshots of mut3 and mut4 interacting with model membranes of various phospholipid compositions (A–F). Color code: phosphorus atom: yellow, POPC black (body) and light gray (choline group), POPS brown (body), gold (headgroup), light yellow (amine of the headgroup), and orange (carboxyl of the headgroup). For clarity, only functional groups of headgroups are shown (spheres) in the upper leaflet. mut3 and mut4 are shown as a ‘tube’ colored from blue (N-terminus) to red (C-terminus). Sidechains are shown as sticks with the following color code: positively charged (blue) and nonpolar (light gray). Snapshots in panels (G–L) refer to rare events observed along the trajectories. Data on HB43 peptide were previously published [<a href="#B42-pharmaceutics-14-01089" class="html-bibr">42</a>]. Full article ">

15 pages, 10118 KiB

Open AccessArticle

Leveraging Affinity Interactions to Prolong Drug Delivery of Protein Therapeutics

by Alan B. Dogan, Katherine E. Dabkowski and Horst A. von Recum

Pharmaceutics 2022, 14(5), 1088; https://doi.org/10.3390/pharmaceutics14051088 - 19 May 2022

Cited by 9 | Viewed by 2597

Abstract

While peptide and protein therapeutics have made tremendous advances in clinical treatments over the past few decades, they have been largely hindered by their ability to be effectively delivered to patients. While bolus parenteral injections have become standard clinical practice, they are insufficient [...] Read more.

While peptide and protein therapeutics have made tremendous advances in clinical treatments over the past few decades, they have been largely hindered by their ability to be effectively delivered to patients. While bolus parenteral injections have become standard clinical practice, they are insufficient to treat diseases that require sustained, local release of therapeutics. Cyclodextrin-based polymers (pCD) have been utilized as a platform to extend the local delivery of small-molecule hydrophobic drugs by leveraging hydrophobic-driven thermodynamic interactions between pCD and payload to extend its release, which has seen success both in vitro and in vivo. Herein, we proposed the novel synthesis of protein–polymer conjugates that are capped with a “high affinity” adamantane. Using bovine serum albumin as a model protein, and anti-interleukin 10 monoclonal antibodies as a functional example, we outline the synthesis of novel protein–polymer conjugates that, when coupled with cyclodextrin delivery platforms, can maintain a sustained release of up to 65 days without largely sacrificing protein structure/function which has significant clinical applications in local antibody-based treatments for immune diseases, cancers, and diabetes. Full article

(This article belongs to the Special Issue Polymers Enhancing Bioavailability in Drug Delivery)

► Show Figures

Graphical abstract

16 pages, 2554 KiB

Open AccessArticle

In Vitro CRISPR/Cas9 Transfection and Gene-Editing Mediated by Multivalent Cationic Liposome–DNA Complexes

by Diana A. Sousa, Ricardo Gaspar, Celso J. O. Ferreira, Fátima Baltazar, Ligia R. Rodrigues and Bruno F. B. Silva

Pharmaceutics 2022, 14(5), 1087; https://doi.org/10.3390/pharmaceutics14051087 - 19 May 2022

Cited by 15 | Viewed by 4526

Abstract

Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated nuclease 9 (Cas9) gene-editing offers exciting new therapeutic possibilities for disease treatment with a genetic etiology such as cancer, cardiovascular, neuronal, and immune disorders. However, its clinical translation is being hampered by the lack [...] Read more.

Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated nuclease 9 (Cas9) gene-editing offers exciting new therapeutic possibilities for disease treatment with a genetic etiology such as cancer, cardiovascular, neuronal, and immune disorders. However, its clinical translation is being hampered by the lack of safe, versatile, and effective nonviral delivery systems. Herein we report on the preparation and application of two cationic liposome–DNA systems (i.e., lipoplexes) for CRISPR/Cas9 gene delivery. For that purpose, two types of cationic lipids are used (DOTAP, monovalent, and MVL5, multivalent with +5e nominal charge), along with three types of helper lipids (DOPC, DOPE, and monoolein (GMO)). We demonstrated that plasmids encoding Cas9 and single-guide RNA (sgRNA), which are typically hard to transfect due to their large size (>9 kb), can be successfully transfected into HEK 293T cells via MVL5-based lipoplexes. In contrast, DOTAP-based lipoplexes resulted in very low transfection rates. MVL5-based lipoplexes presented the ability to escape from lysosomes, which may explain the superior transfection efficiency. Regarding gene editing, MVL5-based lipoplexes achieved promising GFP knockout levels, reaching rates of knockout superior to 35% for charge ratios (+/−) of 10. Despite the knockout efficiency being comparable to that of Lipofectamine 3000^® commercial reagent, the non-specific gene knockout is more pronounced in MVL5-based formulations, probably resulting from the considerable cytotoxicity of these formulations. Altogether, these results show that multivalent lipid-based lipoplexes are promising CRISPR/Cas9 plasmid delivery vehicles, which by further optimization and functionalization may become suitable in vivo delivery systems. Full article

(This article belongs to the Special Issue Lipid-Based Nanocarriers for Non-Viral Gene Delivery)

► Show Figures

Figure 1

Figure 1
The experimental approach to study Cas9/sgRNA plasmid transfection and gene knockout. (a) A plasmid encoding both Cas9 and GFP cassettes is encapsulated into multivalent cationic lipid lipoplexes and administered to HEK 293T cells in vitro. The emergence of a green fluorescence signal in the cells, which indirectly indicates Cas9 expression, is detected by flow cytometry and fluorescence microscopy. (b) To measure CRISPR-mediated gene knockout, HEK 293T cells stably expressing GFP are used. A Cas9 expression plasmid containing a sgRNA to target the GFP gene (PX459-sgRNA-GFP) is designed. The depletion of fluorescence signal associated with GFP knockout is detected by flow cytometry and fluorescence microscopy. To distinguish between CRISPR-mediated GFP knockout and non-specific GFP reduction (e.g., caused by cytotoxicity), a similar plasmid without the sgRNA targeting sequence is used. U6: U6 promoter; sgRNA: contains a target sequence; CMV: CMV promoter, Cas9: Cas9 expression cassette; 2A: 2A self-cleaving peptide; GFP: GFP selection marker; Puro: Puromycin selection marker. Full article ">Figure 2
Chemical structure of monovalent cationic lipid DOTAP and multivalent cationic lipid MVL5 (a). Biophysical characterization of monovalent (DOTAP), and multivalent (MVL5) lipoplexes incorporating the PX458 plasmid at CR (+/−) 3 (b–d) and CR (+/−) 10 (e). The average size and zeta potential of DOTAP and MVL5-based lipoplexes were measured in ultrapure nuclease-free Milli-Q water. Data represent at least three independent experiments and are presented as the mean ± SD. Formulations marked with an asterisk showed bimodal distributions fitted with a biexponential decay model. (f) DLS measurements of the hydrodynamic diameter of the MVL5-based lipoplexes incubated with DMEM at 37 °C. Formulations marked with “*” were fitted with a biexponential decay model. Full article ">Figure 3
In vitro transfection and cytotoxicity in HEK 293T cells transfected with the Cas9/sgRNA-GFP plasmid (PX458). The percentage of GFP-positive cells was measured by flow cytometry. The data are expressed as the mean ± SD (n = 3). (a) Transfection efficiency of DOTAP-based lipoplexes at CR (+/−) 3. One-way ANOVA indicates statistically significant differences within the group assessed by Dunnett’s multiple comparisons test and denoted as follows: **** p ≤ 0.0001. (b,c) Transfection efficiency of MVL5-based lipoplexes at a CR (+/−) of 3 and 10, respectively. (d) Fluorescence microscopy images of HEK 293T cells transfected with 2 µg/mL of PX458 plasmid via multivalent CL-DNA complexes. Scale bars correspond to 100 µm. (e,f) Cytotoxicity profile of monovalent (DOTAP) and multivalent (MVL5) lipoplexes, respectively, as evaluated by the MTT assay. HEK 293T cells were transfected with lipoplexes containing 0.1 µg of PX458. Untreated cells were used as a negative control (100% viable cells). The dashed line corresponds to 70% of cell viability. Data are expressed as the mean ± SD (n = 3). Two-way ANOVA indicates statistically significant differences within the group assessed by Sidak’s multiple comparisons test and denoted as follows: **** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.1, and ns p > 0.05. Full article ">Figure 4
Colocalization of DOTAP and MVL5 lipoplexes signal (red) with LysoSensor (lysosome marker, green) after 4 h of transfection. HEK 293T cells were transfected with Texas-Red-DHPE labeled lipoplexes for 4 h at 37 °C, and then stained with LysoSensor to track the lysosomes’ location. Merged files are representative of the colocalization of CL-DNA complexes with lysosomes. Images were obtained by fluorescence microscopy using a 60× and 100× immersion oil objective. Scales bar corresponds to 10 and 20 µm. Full article ">Figure 5
GFP gene disruption assay in HEK 293T-GFP cells, measured 72 h after treatment with CL-DNA complexes and Lipofectamine 3000. (a,b) Total (KT) and non-specific (KNS) gene silencing efficiency of MVL5-based lipoplexes at a CR (+/−) of 3 and 10, respectively. The percentage of GFP-negative cells was measured by flow cytometry. The data are expressed as the mean± SD (n = 2). KT is measured with the PX459-sgRNA-GFP and KNS is measured with the PX459 empty vector. (c) Fluorescence microscopy images of HEK 293T-GFP cells transfected with MVL5-lipoplexes at a CR (+/−) of 10. Scales bars correspond to 100 µm. Full article ">

19 pages, 1927 KiB

Open AccessArticle

Towards Development of a Non-Toxigenic Clostridioides difficile Oral Spore Vaccine against Toxigenic C. difficile

by Jaime Hughes, Carl Aston, Michelle L. Kelly and Ruth Griffin

Pharmaceutics 2022, 14(5), 1086; https://doi.org/10.3390/pharmaceutics14051086 - 19 May 2022

Cited by 10 | Viewed by 2917

Abstract

Clostridioides difficile is an opportunistic gut pathogen which causes severe colitis, leading to significant morbidity and mortality due to its toxins, TcdA and TcdB. Two intra-muscular toxoid vaccines entered Phase III trials and strongly induced toxin-neutralising antibodies systemically but failed to provide local [...] Read more.

Clostridioides difficile is an opportunistic gut pathogen which causes severe colitis, leading to significant morbidity and mortality due to its toxins, TcdA and TcdB. Two intra-muscular toxoid vaccines entered Phase III trials and strongly induced toxin-neutralising antibodies systemically but failed to provide local protection in the colon from primary C. difficile infection (CDI). Alternatively, by immunising orally, the ileum (main immune inductive site) can be directly targeted to confer protection in the large intestine. The gut commensal, non-toxigenic C. difficile (NTCD) was previously tested in animal models as an oral vaccine for natural delivery of an engineered toxin chimera to the small intestine and successfully induced toxin-neutralising antibodies. We investigated whether NTCD could be further exploited to induce antibodies that block the adherence of C. difficile to epithelial cells to target the first stage of pathogenesis. In NTCD strain T7, the colonisation factor, CD0873, and a domain of TcdB were overexpressed. Following oral immunisation of hamsters with spores of recombinant strain, T7-0873 or T7-TcdB, intestinal and systemic responses were investigated. Vaccination with T7-0873 successfully induced intestinal antibodies that significantly reduced adhesion of toxigenic C. difficile to Caco-2 cells, and these responses were mirrored in sera. Additional engineering of NTCD is now warranted to further develop this vaccine. Full article

(This article belongs to the Special Issue Drug Delivery Systems for Oral Immunotherapy)

► Show Figures

Figure 1

Figure 1
The identification of the 115 bp non-coding integration site in the genome of non-toxigenic C. difficile (NTCD) strain T7. (A) The nucleotide sequence of the integration site in T7 (B) Mapping of the integration site in T7 relative to the genome of the TCD reference strain, 630. The site mapped to intergenic sequence 903 nucleotides upstream of tcdR and 231 nucleotides downstream of tcdC. Full article ">Figure 2
Spores of NTCD T7 germinate in the small intestine of hamsters. Spores were incubated in the contents of fluid taken from proximal, mid and distal portions of non-antibiotic-treated hamsters (n = 6) for 1 h at 37 °C anaerobically. Data are presented as percent germination, determined by: (CFU from germinated spores pre-incubated in intestinal fluid/CFU from germinated spores pre-incubated in 0.1% taurocholate) × 100. Error bars represent the standard deviation (SD). The data were analysed by nonparametric ANOVA (Kruskal–Wallis) with Dunn’s uncorrected comparison. Statistical difference p value: ** p = 0.0017. Full article ">Figure 3
Detection of the expression of antigens in recombinant strains of T7 by SDS-PAGE (10% acrylamide) and Western immunoblotting. Whole cell lysates (WCL) and supernatants of strains T7, T7-0873 and T7-TcdB were harvested from BHIS broth cultures at early stationary phase, A600 nm 1.0. Replica gels for both WCLs and supernatants were stained by Coomassie Blue to confirm equal loading of samples. Western immunoblots with anti-TcdB antibody detected TcdB-RBD mainly intracellularly in T7-TcdB. Western immunoblots with anti-CD0873 antibody detected CD0873 in WCLs and supernatants of all strains with significantly enhanced levels in both recombinant strains. Bands were detected by the addition of the appropriate HRP-conjugated secondary antibody and ECL Western Blotting Detection Reagent. Densitometry was carried out in ImageJ. Error bars represent the standard deviation (SD). Data were analysed by a one-way ANOVA with Dunnett’s multiple comparison. Statistical difference p values: * p < 0.05, **** p < 0.0001. Full article ">Figure 4
Intestinal immune responses in hamsters 2 weeks after oral immunisations with 106 spores of strains T7, T7-TcdB and T7-0873: 3 doses, 2 weeks apart. (A) Total sIgA in intestinal lavages diluted 1:2 was measured by an anti-hamster sIgA ELISA kit (MyBioSource). (B) Adherence of C. difficile strain 630 to Caco-2 monolayers after the bacterial cells were pre-incubated with intestinal lavages diluted 1:2. Cell binding was measured by enumerating CFU from washed Caco-2 cells. Error bars represent the standard deviation (SD). The data were analysed by nonparametric ANOVA (Kruskal–Wallis) with Dunn’s uncorrected comparison. Statistical difference p value: * p < 0.05. Full article ">Figure 5
Systemic immune responses in hamsters 2 weeks after oral immunisations with 106 spores of strains T7, T7-TcdB and T7-0873: 3 doses, 2 weeks apart. Sera were diluted 1:10 and quantified for antigen-specific IgG by indirect ELISA: CD0873 (A) and TcdB-RBD (B). Goat anti-hamster IgG highly cross-adsorbed Biotin antibody (1:20,000) and Streptavidin-HRP (1:200) were used for detection. (C) The effect of sera diluted 1:5 in reducing the binding of C. difficile strain 630 to Caco-2 cells. Error bars represent the standard deviation (SD). The data were analysed by nonparametric ANOVA (Kruskal–Wallis) with Dunn’s uncorrected comparison. Statistical difference p values: * p < 0.05, ** p < 0.01. Full article ">

13 pages, 2511 KiB

Open AccessArticle

Needle-Free Jet Injectors and Nanosuspensions: Exploring the Potential of an Unexpected Pair

by Michele Schlich, Luca Casula, Aurora Musa, Rosa Pireddu, Giulia Pitzanti, Maria Cristina Cardia, Donatella Valenti, Salvatore Marceddu, Anna Maria Fadda, Maria Antonietta De Luca, Chiara Sinico and Francesco Lai

Pharmaceutics 2022, 14(5), 1085; https://doi.org/10.3390/pharmaceutics14051085 - 19 May 2022

Cited by 4 | Viewed by 3599

Abstract

Needle-free liquid jet injectors are medical devices used to administer pharmaceutical solutions through the skin. Jet injectors generate a high-speed stream of liquid medication that can puncture the skin and deliver the drug to the underlying tissues. In this work, we investigated the [...] Read more.

Needle-free liquid jet injectors are medical devices used to administer pharmaceutical solutions through the skin. Jet injectors generate a high-speed stream of liquid medication that can puncture the skin and deliver the drug to the underlying tissues. In this work, we investigated the feasibility of using liquid jet injectors to administer nanosuspensions, assessing the impact of the jet injection on their pharmaceutical and physicochemical properties. For this purpose, the model drug diclofenac was used to prepare a set of nanosuspensions, stabilized by poloxamer 188, and equilibrated at different pHs. The hydrodynamic diameter and morphology of the nanocrystals were analyzed before and after the jet injection across porcine skin in vitro, together with the solubility and release kinetics of diclofenac in a simulated subcutaneous environment. The efficacy of the jet injection (i.e., the amount of drug delivered across the skin) was evaluated for the nanosuspension and for a solution, which was used as a control. Finally, the nanosuspension was administered to rats by jet injector, and the plasma profile of diclofenac was evaluated and compared to the one obtained by jet injecting a solution with an equal concentration. The nanosuspension features were maintained after the jet injection in vitro, suggesting that no structural changes occur upon high-speed impact with the skin. Accordingly, in vivo studies demonstrated the feasibility of jet injecting a nanosuspension, reaching relevant plasma concentration of the drug. Overall, needle-free jet injectors proved to be a suitable alternative to conventional syringes for the administration of nanosuspensions. Full article

(This article belongs to the Special Issue Skin Drug Delivery: Local and Systemic Applications)

► Show Figures

Graphical abstract

Graphical abstract
Full article ">Figure 1
(A) Schematic of the preparation method of DCF NS. (B) Z average (full bars) and PDI (dotted lines) of DCF NS after pH adjustment at the value reported in the x axis. Values represent the average ± standard deviations of n = 3 replicates. Full article ">Figure 2
Z average (full bars) and PDI (dotted lines) of DCF NS before and after jet injection across a skin specimen. Values represent the average ± standard deviations of n = 3 replicates. Pairs labelled with * indicate a difference with p < 0.05. Difference between unlabeled pairs have p > 0.05. Full article ">Figure 3
Scanning electron microscopy analysis of DCF NS (pH 6.0) before (A,B) and after (C,D) the jet injection across a skin specimen. Full article ">Figure 4
Saturation solubility of DCF NS in deionized water at 37 °C before and after the jet injection across a skin specimen. Bars represent the average ± standard deviations of n = 3 independent measurements. Full article ">Figure 5
Drug release from the DCF nanocrystals before or after jet injection in a gel matrix simulating the subcutaneous tissue. Points represent the average of n = 3 independent experimental determinations. Full article ">Figure 6
Scanning electron microscopy analysis of newborn pig skin after the jet injection of a DCF sodium solution (A,B) or DCF NS (pH 6.0) (C,D). Full article ">Figure 7
Concentration versus time curves of plasmatic DCF levels. Results are shown as mean ± SEM of the changes in DCF plasma concentrations after administration of DCF sodium solution (blue circles, n = 4) or DCF NS (grey squares, n = 5). Results analyzed by two-way ANOVA; Bonferroni’s post hoc test. Full article ">

11 pages, 1502 KiB

Open AccessReview

BUB3, beyond the Simple Role of Partner

by Patrícia M. A. Silva and Hassan Bousbaa

Pharmaceutics 2022, 14(5), 1084; https://doi.org/10.3390/pharmaceutics14051084 - 18 May 2022

Cited by 11 | Viewed by 3405

Abstract

The BUB3 protein plays a key role in the activation of the spindle assembly checkpoint (SAC), a ubiquitous surveillance mechanism that ensures the fidelity of chromosome segregation in mitosis and, consequently, prevents chromosome mis-segregation and aneuploidy. Besides its role in SAC signaling, BUB3 [...] Read more.

The BUB3 protein plays a key role in the activation of the spindle assembly checkpoint (SAC), a ubiquitous surveillance mechanism that ensures the fidelity of chromosome segregation in mitosis and, consequently, prevents chromosome mis-segregation and aneuploidy. Besides its role in SAC signaling, BUB3 regulates chromosome attachment to the spindle microtubules. It is also involved in telomere replication and maintenance. Deficiency of the BUB3 gene has been closely linked to premature aging. Upregulation of the BUB3 gene has been found in a variety of human cancers and is associated with poor prognoses. Here, we review the structure and functions of BUB3 in mitosis, its expression in cancer and association with survival prognoses, and its potential as an anticancer target. Full article

(This article belongs to the Special Issue Novel Anticancer Strategies (Volume II))

► Show Figures

Figure 1

Figure 1
Spindle assembly checkpoint mechanism. In response to unattached or improperly attached kinetochores (prometaphase), the SAC is turned ON and promotes the assembly of the mitotic checkpoint complex (MCC), made of MAD2, BUB3, BUBR1 and CDC20. At these kinetochores, the SAC kinase MPS1 recruits BUB3, BUB1 and BUBR1. The MCC inhibits the activity of anaphase-promoting complex/cyclosome (APC/C), leading to the stabilization of separase/securin and CDK1/cyclin B complexes and, consequently, mitotic arrest. The Aurora B kinase (AUR B), associated with centromere heterochromatin, promotes proper kinetochore–microtubule attachments. Once all chromosomes are properly attached to spindle microtubules and are aligned at metaphase plate (metaphase), the SAC is turned OFF, through MCC disassembly, and, consequently, CDC20 can bind and activate the APC/C, resulting in ubiquitylation (ub) of cyclin B and securin mitotic subtracts. In turn, separase can cleave cohesins to promote sister chromatid separation (anaphase), while CDK1 inactivation promotes exit from mitosis. Reprinted from [<a href="#B23-pharmaceutics-14-01084" class="html-bibr">23</a>], MDPI 2021. Full article ">Figure 2
Top overview of Bub3 bound to GLEBS motif determined in yeast. (A) Top view of Bub3 bound to the GLEBS motif from Mad3 (BUBR1 in higher eukaryotes). The GLEBS peptide is colored in gray and lies along the top face of the propeller. N and C termini are labeled, and breaks in main-chain density are denoted with asterisks (*). (B) Top view of Bub3 bound to GLEBS motif from Bub1. Overall, the structures are quite similar, except that the Bub1 GLEBS motif has a shorter loop between helices α1 and α2. (C,D) Side views of Bub3 bound to Mad3/BUBR1 (C) and Bub1 (D) GLEBS motifs. The three-stranded β-sheet that includes the DA loop between blades 5 and 6 of Bub3 projects leftward in these views. Numbers indicate blades. Reprinted with permission from [<a href="#B24-pharmaceutics-14-01084" class="html-bibr">24</a>]. Copyright 2021, National Academy of Sciences, U.S.A. (E) The conserved GLEBS motif in BUB1 and BUBR1 that binds BUB3; alignment of GLEBS motifs from hNUP98, scMad3, scBub1, hBUBR1, mBUB1, and hBuGZ; the completely conserved amino acids, EE, are boxed. Reprinted with permission from [<a href="#B37-pharmaceutics-14-01084" class="html-bibr">37</a>]. Copyright 2022, Elsevier. Full article ">Figure 3
Pan-cancer view of expression of BUB3 protein across cancers. (Upper panel) Comparison between normal (blue) and primary tumors (red); (lower panel) Comparison between TP53-mutant (red) and TP53-non-mutant (orange) tumor samples. RCC: renal cell carcinoma; UCEC: Uterine corpus endometrial carcinoma; CPTAC: Clinical Proteomic Tumor Analysis Consortium. Data were retrieved from UALCAN portal (<a href="http://ualcan.path.uab.edu/index.html" target="_blank">http://ualcan.path.uab.edu/index.html</a>) on 24 December 2021. Full article ">

Journal Menu

Journal Browser

Pharmaceutics, Volume 14, Issue 5 (May 2022) – 226 articles

Further Information

Guidelines

MDPI Initiatives

Follow MDPI