Pharmaceutics

32 pages, 23622 KiB

Open AccessReview

Optimizing Spray-Dried Porous Particles for High Dose Delivery with a Portable Dry Powder Inhaler

by Yoen-Ju Son, Danforth P. Miller and Jeffry G. Weers

Pharmaceutics 2021, 13(9), 1528; https://doi.org/10.3390/pharmaceutics13091528 - 21 Sep 2021

Cited by 16 | Viewed by 4968

This manuscript critically reviews the design and delivery of spray-dried particles for the achievement of high total lung doses (

T L D

) with a portable dry powder inhaler. We introduce a new metric termed the product density, which is simply the [...] Read more.

This manuscript critically reviews the design and delivery of spray-dried particles for the achievement of high total lung doses (

T L D

) with a portable dry powder inhaler. We introduce a new metric termed the product density, which is simply the

T L D

of a drug divided by the volume of the receptacle it is contained within. The product density is given by the product of three terms: the packing density (the mass of powder divided by the volume of the receptacle), the drug loading (the mass of drug divided by the mass of powder), and the aerosol performance (the

T L D

divided by the mass of drug). This manuscript discusses strategies for maximizing each of these terms. Spray drying at low drying rates with small amounts of a shell-forming excipient (low Peclet number) leads to the formation of higher density particles with high packing densities. This enables ultrahigh

T L D

(>100 mg of drug) to be achieved from a single receptacle. The emptying of powder from capsules is directly proportional to the mass of powder in the receptacle, requiring an inhaled volume of about 1 L for fill masses between 40 and 50 mg and up to 3.2 L for a fill mass of 150 mg. Full article

(This article belongs to the Special Issue Inhaled Treatment of Respiratory Infections)

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Figure 1

Figure 1
The unit dose Podhaler DPI and a month’s supply of 240 capsules. Full article ">Figure 2
The total lung dose, <math display="inline"><semantics> <mrow> <mi>T</mi> <mi>L</mi> <mi>D</mi> <mo> </mo> </mrow> </semantics></math> (*) of various inhaled drugs. The <math display="inline"><semantics> <mrow> <mi>T</mi> <mi>L</mi> <mi>D</mi> </mrow> </semantics></math> is divided into low, moderate, high, and ultrahigh dose categories. Abbreviations: ICS, inhaled corticosteroid; LABA, long-acting beta-agonist; LAMA, long-acting muscarinic antagonist; SABA, short-acting beta-agonist; ABIP, amphotericin B inhalation powder (Nektar Therapeutics, San Francisco, CA, USA); CIP, Ciprofloxacin DPI (Bayer, Leverkusen, Germany); TIP, TOBI Podhaler (Novartis, Basel, Switzerland). Full article ">Figure 3
Scanning electron microscopy images of anti-TSLP Fab (CSJ117) particles spray dried with increasing Peclet numbers: (a) smooth spheres with 0% trileucine (Lot A7), (b) dimpled spheres with 2.5% trileucine (Lot A1), (c) corrugated spheres with 15% trileucine (Lot A13) [<a href="#B93-pharmaceutics-13-01528" class="html-bibr">93</a>]. Full article ">Figure 4
Scanning electron micrograph image of compressed spherical anti-TSLP Fab particles (Lot A7) showing large voids in powder bed [<a href="#B93-pharmaceutics-13-01528" class="html-bibr">93</a>]. Full article ">Figure 5
TLD as a function of the product density for various sized capsules. Full article ">Figure 6
(A) Diagram of laser photometer system utilized to determine inhaled volume required to empty powder from a DPI [<a href="#B127-pharmaceutics-13-01528" class="html-bibr">127</a>,<a href="#B128-pharmaceutics-13-01528" class="html-bibr">128</a>]. The diagram was reproduced from [<a href="#B127-pharmaceutics-13-01528" class="html-bibr">127</a>] with permission from Elsevier, 2016; (B) Plot of a representative inspiratory flow profile and corresponding powder emptying profile for TOBI Podhaler using the laser photometer system; (C) Plot of the inhaled volume required to empty a fill mass for various inhaled products. DPIs included in the plot are: Asmanex® Twisthaler®, Pulmicort® Flexhaler®, Spiriva HandiHaler, Advair® Diskus®, Onbrez Breezhaler, indacaterol Simoon™, vardenafil AOS®, Cipro T-326, TOBI Podhaler, CSJ117 T-326 [<a href="#B93-pharmaceutics-13-01528" class="html-bibr">93</a>,<a href="#B127-pharmaceutics-13-01528" class="html-bibr">127</a>,<a href="#B128-pharmaceutics-13-01528" class="html-bibr">128</a>]. It should be noted that the curve is dominated by capsule DPIs except at low fill masses. Full article ">Figure 7
Impact of increases in nominal dose on the incidence of post-inhalation cough. The results are also presented with respect to the various delivery systems (square: nebulizers and soft mist inhalers (SMIs); circle: pressurized metered dose inhalers (pMDIs); triangle: dry powder inhalers (DPIs)). Reproduced with permission from [<a href="#B12-pharmaceutics-13-01528" class="html-bibr">12</a>]. Copyright Elsevier, 2020. Full article ">

20 pages, 6611 KiB

Open AccessArticle

Multifunctional Nanofibrous Dressing with Antimicrobial and Anti-Inflammatory Properties Prepared by Needle-Free Electrospinning

by Laura Victoria Schulte-Werning, Anjanah Murugaiah, Bhupender Singh, Mona Johannessen, Rolf Einar Engstad, Nataša Škalko-Basnet and Ann Mari Holsæter

Pharmaceutics 2021, 13(9), 1527; https://doi.org/10.3390/pharmaceutics13091527 - 21 Sep 2021

Cited by 17 | Viewed by 3743

Abstract

An active wound dressing should address the main goals in wound treatment, which are improved wound healing and reduced infection rates. We developed novel multifunctional nanofibrous wound dressings with three active ingredients: chloramphenicol (CAM), beta-glucan (βG) and chitosan (CHI), of which βG and [...] Read more.

An active wound dressing should address the main goals in wound treatment, which are improved wound healing and reduced infection rates. We developed novel multifunctional nanofibrous wound dressings with three active ingredients: chloramphenicol (CAM), beta-glucan (βG) and chitosan (CHI), of which βG and CHI are active nanofiber-forming biopolymers isolated from the cell walls of Saccharomyces cerevisiae and from shrimp shells, respectively. To evaluate the effect of each active ingredient on the nanofibers’ morphological features and bioactivity, nanofibers with both βG and CHI, only βG, only CHI and only copolymers, polyethylene oxide (PEO) and hydroxypropylmethylcellulose (HPMC) were fabricated. All four nanofiber formulations were also prepared with 1% CAM. The needle-free Nanospider^TM technique allowed for the successful production of defect-free nanofibers containing all three active ingredients. The CAM-containing nanofibers had a burst CAM-release and a high absorption capacity. Nanofibers with all active ingredients (βG, CHI and CAM) showed a concentration-dependent anti-inflammatory activity, while maintaining the antimicrobial activity of CAM. The promising anti-inflammatory properties, together with the high absorption capacity and antimicrobial effect, make these multifunctional nanofibers promising as dressings in local treatment of infected and exuding wounds, such as burn wounds. Full article

(This article belongs to the Special Issue Nanomedicine as a Tool to Improve the Local Delivery of Active Molecules)

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Graphical abstract

Graphical abstract
Full article ">Figure 1
Representative SEM images for each nanofiber formulation. Fiber diameter distribution was determined by measurement of 300 single-fiber diameters for each batch. The overall mean diameter can be found above the diameter distribution (n = 3). (A): βG-CHI-nf, (B): βG-CHI-CAM-nf, (C): βG-nf, (D): βG-CAM-nf, (E): CHI-nf, (F): CHI-CAM-nf, (G): Copol-nf, (H): Copol-CAM-nf. Abbreviations: βG (β-glucan), CHI (chitosan), Copol (co-polymers: polyethylene oxide and hydroxypropylmethylcellulose), CAM (chloramphenicol), nf (nanofiber). Full article ">Figure 2
Cumulative release (%) of chloramphenicol from nanofibers during a 6 h test-period in a Franz diffusion setup. Abbreviations: βG (β-glucan), CHI (chitosan), Copol (co-polymers: polyethylene oxide and hydroxypropylmethylcellulose), CAM (chloramphenicol), nf (nanofibers). Results are presented as mean ± SD (n = 3). Full article ">Figure 3
Relative cell viability (%) of (A) HaCaT cells and (B) macrophages (RAW 264.7) after 24 h incubation at 37 °C and exposure to nanofibers (dissolved in concentrations of 125, 250 and 1000 µg/mL) and chloramphenicol (CAM) (in concentrations of 1.25, 2.5 and 10 µg/mL). Results are presented as mean ± SD (n = 3). Abbreviations: βG (β-glucan), CHI (chitosan), Copol (co-polymers: polyethylene oxide and hydroxypropylmethylcellulose), CAM (chloramphenicol), nf (nanofiber). Full article ">Figure 4
Antibacterial activity of chloramphenicol-containing nanofibers (containing 30 µg of chloramphenicol per fiber) compared to a standard 30 µg chloramphenicol disc as positive control and no-CAM containing nanofibers as negative control (*). Results are expressed as the mean inhibition zone (mm) ± SD (n = 3). Abbreviations: βG (β-glucan), CHI (chitosan), Copol (co-polymers: polyethylene oxide and hydroxypropylmethylcellulose), CAM (chloramphenicol), nf (nanofiber). Full article ">Figure 5
NO production (%) of LPS-induced macrophages (RAW 264.7 cells) after 24 h exposure to nanofibers in three different concentrations (12.5, 25 and 100 µg/mL) compared to untreated cells. Abbreviations: βG (β-glucan), CHI (chitosan), Copol (co-polymers: polyethylene oxide and hydroxypropylmethylcellulose), CAM (chloramphenicol), nf (nanofiber). Results are presented as mean ± SD (n = 3). Formulations marked with * are statistically significant (p < 0.05) compared to untreated LPS-stimulated macrophages. Full article ">

18 pages, 3158 KiB

Open AccessArticle

Endothelial-Derived Extracellular Vesicles Induce Cerebrovascular Dysfunction in Inflammation

by David Roig-Carles, Eduard Willms, Ruud D. Fontijn, Sarai Martinez-Pacheco, Imre Mäger, Helga E. de Vries, Mark Hirst, Basil Sharrack, David K. Male, Cheryl A. Hawkes and Ignacio A. Romero

Pharmaceutics 2021, 13(9), 1525; https://doi.org/10.3390/pharmaceutics13091525 - 21 Sep 2021

Cited by 20 | Viewed by 4203

Abstract

Blood–brain barrier (BBB) dysfunction is a key hallmark in the pathology of many neuroinflammatory disorders. Extracellular vesicles (EVs) are lipid membrane-enclosed carriers of molecular cargo that are involved in cell-to-cell communication. Circulating endothelial EVs are increased in the plasma of patients with neurological [...] Read more.

Blood–brain barrier (BBB) dysfunction is a key hallmark in the pathology of many neuroinflammatory disorders. Extracellular vesicles (EVs) are lipid membrane-enclosed carriers of molecular cargo that are involved in cell-to-cell communication. Circulating endothelial EVs are increased in the plasma of patients with neurological disorders, and immune cell-derived EVs are known to modulate cerebrovascular functions. However, little is known about whether brain endothelial cell (BEC)-derived EVs themselves contribute to BBB dysfunction. Human cerebral microvascular cells (hCMEC/D3) were treated with TNFα and IFNy, and the EVs were isolated and characterised. The effect of EVs on BBB transendothelial resistance (TEER) and leukocyte adhesion in hCMEC/D3 cells was measured by electric substrate cell-substrate impedance sensing and the flow-based T-cell adhesion assay. EV-induced molecular changes in recipient hCMEC/D3 cells were analysed by RT-qPCR and Western blotting. A stimulation of naïve hCMEC/D3 cells with small EVs (sEVs) reduced the TEER and increased the shear-resistant T-cell adhesion. The levels of microRNA-155, VCAM1 and ICAM1 were increased in sEV-treated hCMEC/D3 cells. Blocking the expression of VCAM1, but not of ICAM1, prevented sEV-mediated T-cell adhesion to brain endothelia. These results suggest that sEVs derived from inflamed BECs promote cerebrovascular dysfunction. These findings may provide new insights into the mechanisms involving neuroinflammatory disorders. Full article

(This article belongs to the Special Issue Biological Barriers in Health and Disease)

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Figure 1

Figure 1
Characterisation of extracellular vesicles in inflammation. (a) Graphic diagram of the isolation of extracellular vesicles (EVs) both small (sEVs) and large (L-EVs) from untreated hCMEC/D3 cells (quiescent sEVs or L-EVs) and those incubated with 10-ng/mL TNFα and IFNy (cytokine-sEVs or -L-EVs) for 24 h using a combination of ultracentrifugation (dUC) and filtration steps. (b) Nanoparticle tracking analysis (NTA) measurements of isolated EVs revealed significantly increased numbers of cytokine-sEVs after the cytokine treatment compared to the controls. (c) Size distribution measured by NTA showed that most isolated EVs were smaller than 200 nm in all EV subsets (left image shows sEVs, whereas the right image shows L-EVs). (d) Representative images of cytokine-sEVs and L-EVs from the Nanoparticle Tracking Analysis (e) The table summarises the mean; mode and D10, D50 and D90 diameter sizes of sEVs and L-EVS. (f). Transmission electron microscopy (TEM) images of cytokine-sEVs and cytokine-L-EVs. Arrows point to sEVs in both images. (g) Immunoblots for CD9, CD63 and HSP70 in cytokine-sEVs (sEV) and cell lysate (CL) controls. (h) Concentrations of TNFα and IFNy in the cytokine-sEV fractions and cell-conditioned medium (CCM). Data are shown as the mean ± SEM, n = 2 (d), n = 3 (f) and n = 4 (b,c). The means were compared with one-way ANOVA, followed by Tukey’s post-hoc test. * p < 0.05, ## or ** p < 0.01, *** p < 0.001 and **** p < 0.0001. Full article ">Figure 2
Characterisation of the uptake of cytokine-sEVs by naïve brain endothelium. (a) Confocal images of the uptake of 0.5 × 108 DiO-labelled cytokine-sEVs/µL by human microvascular brain endothelial cells (hCMEC/D3 cells) at different time points (0, 6, 24 and 48 h). (b) Flow cytometry measurements of DIO-labelled cytokine-sEVs incubated with hCMEC/D3. The graph shows a quantification of the median fluorescent intensity (MFI) of the uptake of cytokine-sEVs (0.1, 0.5 and 1×108 sEVs/µL) by naïve hCMEC/D3 cells. Data is shown as the mean ± SEM (n = 3). The means were compared with one-way ANOVA followed by Tukey’s multiple comparison test. * p < 0.05 and *** p < 0.001. Full article ">Figure 3
Cytokine-sEVs decrease the transendothelial resistance of hCMEC/D3 cells. (a) TEER values following hCMEC/D3 cells with varying concentrations of cytokine-derived small EVs (cytokine-sEVs) (0.1, 0.5 and 1 × 108 sEVs/µL). Data are shown as the percentage fold change of treated relative to untreated cells at each time point. (b) Comparison of the effects of 0.5 × 108 sEVs/µL cytokine-sEVs and quiescent sEVs on the TEER values. (c) Para-cellular permeability experiment of 70-KDa FITC-Dextran after a 0.1 × 108 cytokine-sEVs/ul stimulation for 6, 24 and 48 h. Data is shown as the mean ± SEM. The means are compared by two-way ANOVA followed by Tukey’s post-hoc for multiple comparisons, n = 3 (b) and 4 (a). * p < 0.05, ** p < 0.01, *** and p < 0.001 compared to untreated cells. Full article ">Figure 4
Cytokine-sEVs increase Jurkat T-cell adhesion to the brain endothelium. (a) Representative brightfield (top) and fluorescent (FITC, bottom) images of Jurkat T-cell adhesion (round-shaped cells) to hCMEC/D3 cells (spindle-shaped cells) under flow conditions in their absence (control, left panels), with the addition of cytokine-derived small EVs (cytokine-sEVs) (middle panels) or cytokines (1-ng/mL TNFα + IFNy, right panels). (b) Quantification of the number of firmly adhered T cells per field of view (FAJCN) (FOV 640 × 480 μm) following incubation with increasing doses of cytokine-sEVs (0.1, 0.5 and 1 × 108 cytokine-sEVs/µL) for 24 h. (c) Time–course analysis (6, 24 and 48 h) of the effect of 1 × 108 cytokine-sEVs on FAJCN on naïve endothelium. (d) Comparison of cytokine-sEVs and quiescent sEVs (0.5 × 108 sEVs/µL for 24 h) on FACJN in naïve hCMEC/D3 cells. Data is shown as the mean ± SEM, and n = 4 (b,c) and 3 (d). The means are compared by one-way ANOVA followed by Dunnett’s post-hoc for multiple comparisons, * p < 0.05 and ** p < 0.01 and *** p < 0.001 to the untreated condition. Full article ">Figure 5
Characterisation of inflammation-related miRNAs and mRNAs in sEV and secreting hCMEC/D3 cells. (a) Quantification of levels of miRNA-155-5p, miRNA-146a, miRNA-146b, miRNA-126-5p, miRNA-126-3p and miRNA-24 in cytokine-sEVs and quiescent sEVs. (b) Levels of miRNA-155-5p in naïve hCMEC/D3 cells and those treated with 10-ng/mL TNFα and IFNy. (c). mRNA levels of vascular cell adhesion molecule (VCAM1), intercellular adhesion molecule (ICAM1) and carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) in cytokine-sEVs and quiescent sEVs. (d) Levels of VCAM1 and ICAM1 mRNA in naïve hCMEC/D3 cells and those treated with 10-ng/mL TNFα and IFNy. Data are shown as the mean ± SEM (n = 3). The means were compared by unpaired two-tailed t-tests, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 compared to quiescent sEV cargos. Full article ">Figure 6
Cytokine-sEVs induce a proinflammatory profile in recipient hCMEC/D3 cells. (a) Relative levels of miRNA-155-5p, miRNA-126-3p and miRNA-24 in hCMEC/D3 cells after treatment with cytokine- or quiescent sEVs. (b) Relative levels of ICAM1, VCAM1, occludin (OCLDN) and claudin-5 (CLDN5) on hCMEC/D3 cells treated with cytokine- or quiescent sEVs for 24 h. (c,d) Immunoblot and quantification of cytokine-sEV-treated or untreated (control) hCMEC/D3 cells for ICAM1 and VCAM1. (e) Effect of blocking of ICAM and VCAM alone and in combination with the adhesion of Jurkat T cells to untreated or cytokine-sEV-treated hCMEC/D3 cells. Data is shown as the mean ± SEM, n = 3 (a,b,d) and n = 4 (e). The means were compared by two-way ANOVA followed by Tukey’s comparison test (a,b) or paired two-tailed t-test (d), * p < 0.05, ** p < 0.01 and *** p < 0.001 in comparison to untreated cell levels (a,b,d) or to the IgG control (e). Full article ">Figure 7
The proposed model for the cytokine-sEV role in modulating cerebrovascular functions. (1) The incubation of hCMEC/D3 cells with 10 ng/mL of TNFα and IFNy increased the secretion of small EVs (cytokine-sEVs). (2) These cytokine-sEVs carried elevated levels of the proinflammatory miRNA-155, as well as mRNAs of VCAM1 and ICAM1. (3) Naïve hCMEC/D3 cells take up cytokine-sEVs, leading to increased intracellular levels of miRNA-155 and ICAM1 and VCAM1 in recipient hCMEC/D3 cells (4). Consequently, the endothelial resistance is decreased (5), while the firm adhesion of Jurkat T cells (6) is increased in recipient endothelial cells. Full article ">

27 pages, 6328 KiB

Open AccessReview

3D Printing of Thermo-Sensitive Drugs

by Sadikalmahdi Abdella, Souha H. Youssef, Franklin Afinjuomo, Yunmei Song, Paris Fouladian, Richard Upton and Sanjay Garg

Pharmaceutics 2021, 13(9), 1524; https://doi.org/10.3390/pharmaceutics13091524 - 21 Sep 2021

Cited by 34 | Viewed by 6684

Abstract

Three-dimensional (3D) printing is among the rapidly evolving technologies with applications in many sectors. The pharmaceutical industry is no exception, and the approval of the first 3D-printed tablet (Spiratam^®) marked a revolution in the field. Several studies reported the fabrication of [...] Read more.

Three-dimensional (3D) printing is among the rapidly evolving technologies with applications in many sectors. The pharmaceutical industry is no exception, and the approval of the first 3D-printed tablet (Spiratam^®) marked a revolution in the field. Several studies reported the fabrication of different dosage forms using a range of 3D printing techniques. Thermosensitive drugs compose a considerable segment of available medications in the market requiring strict temperature control during processing to ensure their efficacy and safety. Heating involved in some of the 3D printing technologies raises concerns regarding the feasibility of the techniques for printing thermolabile drugs. Studies reported that semi-solid extrusion (SSE) is the commonly used printing technique to fabricate thermosensitive drugs. Digital light processing (DLP), binder jetting (BJ), and stereolithography (SLA) can also be used for the fabrication of thermosensitive drugs as they do not involve heating elements. Nonetheless, degradation of some drugs by light source used in the techniques was reported. Interestingly, fused deposition modelling (FDM) coupled with filling techniques offered protection against thermal degradation. Concepts such as selection of low melting point polymers, adjustment of printing parameters, and coupling of more than one printing technique were exploited in printing thermosensitive drugs. This systematic review presents challenges, 3DP procedures, and future directions of 3D printing of thermo-sensitive formulations. Full article

(This article belongs to the Special Issue Printed Pharmaceuticals in Future Healthcare)

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10 pages, 1804 KiB

Open AccessArticle

Tablet Splitting in Elderly Patients with Dementia: The Case of Quetiapine

by Roberta Ganzetti, Serena Logrippo, Matteo Sestili, Alessandro Caraffa, Marco Cespi, Giuseppe Pelliccioni, Paolo Blasi and Giulia Bonacucina

Pharmaceutics 2021, 13(9), 1523; https://doi.org/10.3390/pharmaceutics13091523 - 20 Sep 2021

Cited by 7 | Viewed by 5438

Abstract

Quetiapine is an atypical antipsychotic approved for treating schizophrenia, bipolar depression, and mania but is frequently used in an off-label manner to control the behavioral and psychological symptoms of dementia in elderly patients with dementia. Due to the need to personalize doses for [...] Read more.

Quetiapine is an atypical antipsychotic approved for treating schizophrenia, bipolar depression, and mania but is frequently used in an off-label manner to control the behavioral and psychological symptoms of dementia in elderly patients with dementia. Due to the need to personalize doses for elderly patients with dementia, quetiapine tablet manipulation is widespread in hospital settings, long-term care facilities, and patient homes. The aim of this study was to assess the impact of the different splitting techniques on quetiapine fumarate tablets by analysing the obtained sub-divided tablets and to discuss compliance with the European Pharmacopoeia limits on whole and split tablets. Quetiapine fumarate tablets of two dose strengths were taken at random (in a number able to assure a power of 0.8 during statistical comparison) and were split with a kitchen knife or tablet cutter. The weight and the drug content were determined for each half tablet. The obtained data were compared to the European Pharmacopoeia limits. The differences between the different splitting techniques were statistically tested. Data showed that split tablets, independently of the dose strength and the technique employed, were not compliant with the European Pharmacopoeia specifications for both entire and subdivided tablets in terms of weight and content uniformity. Thus, such a common practice could have potential effects on treatment efficacy and toxicity, especially when also considering the fragility of the elderly target population in which polypharmacotherapy is very common. These results indicate a compelling need for flexible quetiapine formulations that can assure more accurate dose personalization. Full article

(This article belongs to the Special Issue Advance in Development of Patient-Centric Dosage Form)

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Graphical abstract

Graphical abstract
Full article ">Figure 1
Frequency of tablet splitting techniques reported by patients and/or family caregivers. Full article ">Figure 2
Effect of tablet splitting on weight for 100 mg and 25 mg quetiapine tablets. The control groups are represented by the half of the whole tablet weights. The central black lines represent the mean values, while the whiskers represent the standard deviations. The solid grey lines and the dashed grey lines in the background represent the European Pharmacopoeia limits for the uniformity of mass of single-dose preparations (quetiapine 100 mg: solid grey lines ± 15% and dash grey lines ± 7.5%; quetiapine 25 mg: solid grey lines ± 20% and dash grey lines ± 10%) For simplicity, the European Pharmacopoeia limits showed in the plots have been calculated on the mean weight values of the control groups. Full article ">Figure 3
Effect of tablet splitting on drug content for 100 mg and 25 mg quetiapine tablets. The control groups are represented by the half drug content of the whole tablet contents. The central black lines represent the mean values, while the whiskers the standard deviations. The solid grey lines and the dashed grey lines in the background represent the European Pharmacopoeia limits for the uniformity of content of single-dose preparations: solid grey lines ± 25%, and dash grey lines ± 15%. For simplicity, the European Pharmacopoeia limits showed in the plots have been calculated on the mean drug content values of the control groups. Full article ">Figure 4
Pearson correlation analysis between weight and drug content for all the three the groups of half tablets: control (the values refer to the half weight or half content of the whole tablets), tablet cutter, and knife. Full article ">

13 pages, 11708 KiB

Open AccessReview

Selinexor and the Selective Inhibition of Nuclear Export: A New Perspective on the Treatment of Sarcomas and Other Solid and Non-Solid Tumors

by Antonella Lucia Marretta, Giuseppe Di Lorenzo, Dario Ribera, Lucia Cannella, Claudia von Arx, Alessandra Bracigliano, Ottavia Clemente, Roberto Tafuto, Antonio Pizzolorusso and Salvatore Tafuto

Pharmaceutics 2021, 13(9), 1522; https://doi.org/10.3390/pharmaceutics13091522 - 20 Sep 2021

Cited by 6 | Viewed by 4461

Abstract

Nucleocytoplasmic transport has been found dysregulated in many types of cancer and is often described as a poor prognostic factor. Specifically, Exportin-1 (XPO1) has been found overexpressed in many tumors and has become an attractive target in molecular oncology and therapeutics development. The [...] Read more.

Nucleocytoplasmic transport has been found dysregulated in many types of cancer and is often described as a poor prognostic factor. Specifically, Exportin-1 (XPO1) has been found overexpressed in many tumors and has become an attractive target in molecular oncology and therapeutics development. The selective inhibitor of nuclear export, Selinexor, is one of the most scientifically interesting drugs that targets XPO1 in clinical development. In this review, we summarized the most relevant preclinical and clinical results achieved for non-solid tumors, sarcomas, and other kind of solid tumors. Full article

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13 pages, 1931 KiB

Open AccessArticle

Polylactide Nanocapsules Attenuate Adverse Cardiac Cellular Effects of Lyso-7, a Pan-PPAR Agonist/Anti-Inflammatory New Thiazolidinedione

by Giani M. Garcia, Jérôme Roy, Ivan R. Pitta, Dulcinéia S. P. Abdalla, Andrea Grabe-Guimarães, Vanessa C. F. Mosqueira and Sylvain Richard

Pharmaceutics 2021, 13(9), 1521; https://doi.org/10.3390/pharmaceutics13091521 - 20 Sep 2021

Cited by 6 | Viewed by 2626

Abstract

Lyso-7 is a novel synthetic thiazolidinedione, which is a receptor (pan) agonist of PPAR α,β/δ,γ with anti-inflammatory activity. We investigated the cardiotoxicity of free Lyso-7 in vitro (4.5–450 nM), and Lyso-7 loaded in polylactic acid nanocapsules (NC) in vivo (Lyso-7-NC, 1.6 mg/kg). In [...] Read more.

Lyso-7 is a novel synthetic thiazolidinedione, which is a receptor (pan) agonist of PPAR α,β/δ,γ with anti-inflammatory activity. We investigated the cardiotoxicity of free Lyso-7 in vitro (4.5–450 nM), and Lyso-7 loaded in polylactic acid nanocapsules (NC) in vivo (Lyso-7-NC, 1.6 mg/kg). In previous work, we characterized Lyso-7-NC. We administered intravenously Lyso-7, Lyso-7-NC, control, and blank-NC once a day for seven days in mice. We assessed cell contraction and intracellular Ca²⁺ transients on single mice cardiomyocytes enzymatically isolated. Lyso-7 reduced cell contraction and accelerated relaxation while lowering diastolic Ca²⁺ and reducing Ca²⁺ transient amplitude. Lyso-7 also promoted abnormal ectopic diastolic Ca²⁺ events, which isoproterenol dramatically enhanced. Incorporation of Lyso-7 in NC attenuated drug effects on cell contraction and prevented its impact on relaxation, diastolic Ca²⁺, Ca²⁺ transient amplitude, Ca²⁺ transient decay kinetics, and promotion of diastolic Ca²⁺ events. Acute effects of Lyso-7 on cardiomyocytes in vitro at high concentrations (450 nM) were globally similar to those observed after repeated administration in vivo. In conclusion, we show evidence for off-target effects of Lyso-7, seen during acute exposure of cardiomyocytes to high concentrations and after repeated treatment in mice. Nano-encapsulation of Lyso-7 in polymeric NC attenuated the unwanted effects, particularly ectopic Ca²⁺ events known to support life-threatening arrhythmias favored by stress or exercise. Full article

(This article belongs to the Special Issue Polymeric Nanocapsules in Drug Delivery)

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Figure 1
Chemical structure of Lyso-7 and schematic representation of polymeric nanocapsules (A). Presentation of the experimental protocol used to treat mice and isolate the cardiomyocytes to test cardiotoxicity of the formulations (B) directly in vitro (acute effects) or after in vivo treatment followed by cardiomyocyte isolation. Full article ">Figure 2
Effect of in vivo administration of Lyso-7 and Lyso-7-NC (1.6 mg/kg, once a day for seven days) on contraction (A–D) and Ca2+ transient (E–H) in freshly isolated cardiomyocytes. Typical recordings of the effects of Lyso-7 (free form) and Lyso-7-NC on sarcomere length (SL) shortening (A) and Ca2+ transient (E), both under field stimulation at 1 Hz. (B–D): averaged data of resting SL, SL shortening and relaxation, respectively, and (F–H): averaged data of diastolic Ca2+, Ca2+ transient and decay of Ca2+ transient (Tau), respectively, all for the four experimental groups (Control, Lyso-7, NC, and Lyso-7-NC). ANOVA followed by Tukey post-test (p < 0.05). * Lyso-7 vs. Control; ! Lyso-7-NC vs. Lyso-7. n = 4; n = 12–18. Full article ">Figure 3
Effect of in vivo administration of Lyso-7 and Lyso-7-NC (1.6 mg/kg, once a day for seven days) on contraction (A–D) and Ca2+ transient (E–H) of freshly isolated cardiomyocytes exposed in vitro to Isoproterenol (ISO, 10 nM). (A–C): averaged data of resting sarcomere length (SL), SL shortening and relaxation, respectively, and (D–F): averaged data of diastolic Ca2+, Ca2+ transient and decay of Ca2+ transient (Tau), respectively, all for the four experimental groups (Control, Lyso-7, NC, and Lyso-7-NC). ANOVA followed by Tukey post-test (p < 0.05). * Lyso-7 vs. Control; ! Lyso-7-NC vs. Lyso-7. n = 4; n = 12–18. Full article ">Figure 4
Effect of in vivo administration of Lyso-7 and Lyso-7-NC (1.6 mg/kg, once a day for seven days) on cardiomyocytes’ ectopic diastolic Ca2+ waves. (A) Representative recordings of Ca2+ waves during resting periods after a train of field stimulation at 1 Hz in cardiomyocytes from the four experimental groups (Control, Lyso-7, NC, and Lyso-7-NC); (B) Representative recordings of Ca2+ waves during resting periods after a train of field stimulation at 1 Hz in cardiomyocytes from the four the same experimental groups treated in vitro with Isoproterenol (ISO, 10 nM); (C,D) percentage of cardiomyocytes developing at least one spontaneous Ca2+ wave in absence and presence of ISO (10 nM) in vitro, respectively, for the four experimental groups (Control, Lyso-7, NC, and Lyso-7-NC). We used a t-test. p < 0.05,* Lyso-7 vs. control; ! Lyso-7-NC vs. Lyso-7. a.u.: arbitrary units. n = 4; n = 12–18. Full article ">Figure 5
Acute effect of Lyso-7 on contraction (A–D) and Ca2+ transient (E–H) in cardiomyocytes of untreated mice. Typical recordings of Lyso-7 (450 nM) effects on sarcomere length (SL) shortening (A) and Ca2+ transient (E), both under field stimulation at 1 Hz. (B–D) averaged data of resting SL, SL shortening and relaxation, respectively, and (F–H): averaged data of diastolic Ca2+, Ca2+ transient and decay of Ca2+ transient (Tau), respectively, for Control, and increasing concentration of Lyso-7 (4.5, 45, and 450 nM). ANOVA followed by Tukey post-test (p < 0.05). * Lyso-7 vs. Control. Control refers to the absence of Lyso-7. n = 12–18. Full article ">Figure 6
Acute in vitro effect of Lyso-7 on contraction (A–D) and Ca2+ transient (E–H) of cardiomyocytes under IsoproTable 10. nM). Typical recordings of Lyso-7 (450 nM) effects on (A) sarcomere length (SL) shortening and (E) Ca2+ transient, both under field stimulation at 1 Hz. (B–D) averaged data of resting SL, SL shortening and relaxation, respectively, and (F–H) averaged data of diastolic Ca2+, Ca2+ transient and decay of Ca2+ transient (Tau), respectively, for Control, and increasing concentration of Lyso-7 (4.5, 45, and 450 nM). ANOVA followed by Tukey post-test (p < 0.05). * Lyso-7 vs. Control. Control refers to the absence of Lyso-7. n = 12–18. Full article ">Figure 7
Acute in vitro effect of Lyso-7 on cardiomyocytes’ abnormal spontaneous diastolic Ca2+ events under IsoproTable 10. nM) challenge. (A) percentage of cardiomyocytes developing at least one spontaneous Ca2+ wave in the absence (left panel) and presence (right panel) of Isoproterenol (ISO, 10 nM) during resting periods after a train of field stimulation at 1 Hz in cardiomyocytes. (B) Representative recordings of Ca2+ waves under ISO. n = 12–18 cells. (C) Typical line-scan confocal images of Ca2+ sparks from Fluo-4-AM loaded cardiomyocytes; n = 7–10. (D) Mean of frequency of Ca2+ sparks measured in the different conditions indicated. C = Control refers to the absence of Lyso-7 (450 nM). We used a t-test. p < 0.05, * Lyso-7 vs. Control in the absence of ISO (left part of the panel); # Lyso-7 vs. Control in the presence of ISO (right part of the panel); ! with ISO vs. without ISO. Full article ">

24 pages, 1600 KiB

Open AccessReview

Non-Viral Gene Delivery Systems for Treatment of Myocardial Infarction: Targeting Strategies and Cardiac Cell Modulation

by Jieting Wang, Luying Yu, Ao Zhou, Jie Liu, Kai Wang, Ying Luo and Fang Wang

Pharmaceutics 2021, 13(9), 1520; https://doi.org/10.3390/pharmaceutics13091520 - 19 Sep 2021

Cited by 6 | Viewed by 3663

Abstract

Cardiovascular diseases (CVD) are the leading cause of morbidity and mortality worldwide. Conventional therapies involving surgery or pharmacological strategies have shown limited therapeutic effects due to a lack of cardiac tissue repair. Gene therapy has opened an avenue for the treatment of cardiac [...] Read more.

Cardiovascular diseases (CVD) are the leading cause of morbidity and mortality worldwide. Conventional therapies involving surgery or pharmacological strategies have shown limited therapeutic effects due to a lack of cardiac tissue repair. Gene therapy has opened an avenue for the treatment of cardiac diseases through manipulating the underlying gene mechanics. Several gene therapies for cardiac diseases have been assessed in clinical trials, while the clinical translation greatly depends on the delivery technologies. Non-viral vectors are attracting much attention due to their safety and facile production compared to viral vectors. In this review, we discuss the recent progress of non-viral gene therapies for the treatment of cardiovascular diseases, with a particular focus on myocardial infarction (MI). Through a summary of delivery strategies with which to target cardiac tissue and different cardiac cells for MI treatment, this review aims to inspire new insights into the design/exploitation of non-viral delivery systems for gene cargos to promote cardiac repair/regeneration. Full article

(This article belongs to the Special Issue Novel Insights in Delivery Systems: Phytochemicals and Biopharmaceuticals)

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17 pages, 3216 KiB

Open AccessArticle

Co-Spray Dried Nafamostat Mesylate with Lecithin and Mannitol as Respirable Microparticles for Targeted Pulmonary Delivery: Pharmacokinetics and Lung Distribution in Rats

by Ji-Hyun Kang, Young-Jin Kim, Min-Seok Yang, Dae Hwan Shin, Dong-Wook Kim, Il Yeong Park and Chun-Woong Park

Pharmaceutics 2021, 13(9), 1519; https://doi.org/10.3390/pharmaceutics13091519 - 19 Sep 2021

Cited by 10 | Viewed by 3828

Abstract

Coronavirus disease 2019 (COVID-19), caused by a new strain of coronavirus called severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is spreading rapidly worldwide. Nafamostat mesylate (NFM) suppresses transmembrane serine protease 2 and SARS-CoV-2 S protein-mediated fusion. In this study, pharmacokinetics and lung distribution [...] Read more.

Coronavirus disease 2019 (COVID-19), caused by a new strain of coronavirus called severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is spreading rapidly worldwide. Nafamostat mesylate (NFM) suppresses transmembrane serine protease 2 and SARS-CoV-2 S protein-mediated fusion. In this study, pharmacokinetics and lung distribution of NFM, administered via intravenous and intratracheal routes, were determined using high performance liquid chromatography analysis of blood plasma, lung lumen using bronchoalveolar lavage fluid, and lung tissue. Intratracheal administration had higher drug delivery and longer residual time in the lung lumen and tissue, which are the main sites of action, than intravenous administration. We confirmed the effect of lecithin as a stabilizer through an ex vivo stability test. Lecithin acts as an inhibitor of carboxylesterase and delays NFM decomposition. We prepared inhalable microparticles with NFM, lecithin, and mannitol via the co-spray method. The formulation prepared using an NFM:lecithin:mannitol ratio of 1:1:100 had a small particle size and excellent aerodynamic performance. Spray dried microparticles containing NFM, lecithin, and mannitol (1:1:100) had the longest residual time in the lung tissue. In conclusion, NFM-inhalable microparticles were prepared and confirmed to be delivered into the respiratory tract, such as lung lumen and lung tissue, through in vitro and in vivo evaluations. Full article

(This article belongs to the Special Issue Therapeutic Formulations of Repurposed Drugs against COVID-19)

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18 pages, 8557 KiB

Open AccessArticle

Mesoporous Calcium-Silicate Nanoparticles Loaded with Low-Dose Triton-100+Ag⁺ to Achieve Both Enhanced Antibacterial Properties and Low Cytotoxicity for Dentin Disinfection of Human Teeth

by Mengting Duan, Wei Fan and Bing Fan

Pharmaceutics 2021, 13(9), 1518; https://doi.org/10.3390/pharmaceutics13091518 - 19 Sep 2021

Cited by 7 | Viewed by 3075

Abstract

Mesoporous calcium-silicate nanoparticles (MCSNs) are excellent biomaterials for controlled drug delivery and mineralization induction. In this study, MCSNs were loaded with low-dose silver ion (Ag⁺) and Triton X-100 (TX-100) as the M-AgTX to achieve both enhanced antibacterial properties and low cytotoxicity [...] Read more.

Mesoporous calcium-silicate nanoparticles (MCSNs) are excellent biomaterials for controlled drug delivery and mineralization induction. In this study, MCSNs were loaded with low-dose silver ion (Ag⁺) and Triton X-100 (TX-100) as the M-AgTX to achieve both enhanced antibacterial properties and low cytotoxicity for dentin disinfection. The physicochemical property, biocompatibility, infiltration ability into dentinal tubules, anti-bacterial ability against both planktonic Enterococcusfaecalis (E. faecalis) and its biofilm on dentin, effects on dentin microhardness and in vitro mineralization property were systematically investigated. Results confirmed that the MCSNs and M-AgTX nanoparticles showed typical morphology of mesoporous materials and exhibited sustained release of chemicals with an alkaline pH value over time. M-AgTX also exhibited excellent biocompatibility on MC3T3-E1 cells and could eliminate 100% planktonic E. faecalis after 48-h treatment. On dentin slices, it could enter dentinal tubules by ultrasonic activation and inhibit the growth of E. faecalis on dentin. M-AgTX could completely inactive 28-day E. faecalis biofilm. TEM confirmed the destruction of cell membrane integrity and Ag⁺ infiltration into bacteria by M-AgTX. Besides, dentin slices medicated with M-AgTX nanoparticles displayed an increased microhardness. After being immersed in SBF for 7 days, apatite crystals could be observed on the surface of the material tablets. M-AgTX could be developed into a new multifunctional intra-canal medication or bone defect filling material for infected bone defects due to its sustained release profile, low cytotoxicity, infiltration ability, enhanced anti-bacterial and mineralization features. Full article

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16 pages, 1978 KiB

Open AccessArticle

Increased Bioavailability of β-Alanine by a Novel Controlled-Release Powder Blend Compared to a Slow-Release Tablet

by Lydia de Salazar, Ignacio Segarra, Francisco Javier López-Román, Antonio Torregrosa-García, Silvia Pérez-Piñero and Vicente Ávila-Gandía

Pharmaceutics 2021, 13(9), 1517; https://doi.org/10.3390/pharmaceutics13091517 - 19 Sep 2021

Cited by 3 | Viewed by 3412

Abstract

Background: β-Alanine is a sport supplement with increasing popularity due to its consistent ability to improve physical performance, with the downside of requiring several weeks of supplementation as imposed to the maximum daily and single dose tolerated without side effects (i.e., paresthesia). To [...] Read more.

Background: β-Alanine is a sport supplement with increasing popularity due to its consistent ability to improve physical performance, with the downside of requiring several weeks of supplementation as imposed to the maximum daily and single dose tolerated without side effects (i.e., paresthesia). To date, the only alternative to overcome this problem has been use of a sustained-release tablet, while powders are the most commonly used format to deliver several grams of amino acids in a single dose. In this study we assessed the bioavailability, pharmacokinetics and paresthesia effect of β-alanine after administration in a novel controlled-released powder blend (test) versus a sustained-release tablet (reference). Methods: Twelve subjects (25.6 ± 3.2 y, 50% female) participated in a randomized, single-blind, crossover study. Each participant was administered orally the test (β-alanine 8 g, l-histidine 300 mg, carnosine 100 mg) or the reference product (10 tablets to reach β-alanine 8 g, Zinc 20 mg) with a 1-week washout period. β-Alanine plasma concentrations (0–8 h) were determined by LC-MS/MS and model-independent pharmacokinetic analysis was carried out. Paresthesia intensity was evaluated using a Visual Analog Score (VAS) and the categorical Intensity Sensory Score (ISS). Results: The C_MAX and AUC₀_→_∞ increased 1.6- and 2.1-fold (both p < 0.001) in the test product, respectively, which yielded 2.1-fold higher bioavailability; K_a decreased in the test (0.0199 ± 0.0107 min⁻¹) versus the reference (0.0299 ± 0.0121 min⁻¹) product (p = 0.0834) as well as V/F and Cl/F (both p < 0.001); MRT₀_→last increased in the test (143 ± 19 min) versus reference (128 ± 16 min) formulation (p = 0.0449); t_1/2 remained similar (test: 63.5 ± 8.7 min, reference: 68.9 ± 9.8 min). Paresthesia E_MAX increased 1.7-fold using the VAS (p = 0.086) and the ISS (p = 0.009). AUEC increased 1.9-fold with the VAS (p = 0.107) and the ISS (p = 0.019) reflecting scale intrinsic differences. Pharmacokinetic-pharmacodynamic analysis showed a clockwise hysteresis loop without prediction ability between C_MAX, AUC₀_→_∞ and E_MAX or AUEC. No side effects were reported (except paresthesia). Conclusions: The novel controlled-release powder blend shows 100% higher bioavailability of β-alanine, opening a new paradigm that shifts from chronic to short or mid-term supplementation strategies to increase carnosine stores in sports nutrition. Full article

(This article belongs to the Section Drug Delivery and Controlled Release)

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14 pages, 1501 KiB

Open AccessArticle

Synthesis and Biochemical Evaluation of Baicalein Prodrugs

by Sang-Hyun Son, Jinhong Kang, Myunghwan Ahn, Soyeon Nam, Yong Woo Jung, Ki Yong Lee, Young Ho Jeon, Youngjoo Byun and Kiho Lee

Pharmaceutics 2021, 13(9), 1516; https://doi.org/10.3390/pharmaceutics13091516 - 19 Sep 2021

Cited by 10 | Viewed by 3712

Abstract

Baicalein (5,6,7-trihydroxy-2-phenyl-4H-1-benzopyran-4-one), a flavonoid analog from Scutellaria baicalensis, possesses several pharmacological activities including antioxidant, antiproliferative, and anti-inflammatory activities. We previously reported that baicalein inhibits the thymic stromal lymphopoietin (TSLP)/TSLP receptor (TSLPR) signaling pathways and can be used as an active ingredient in [...] Read more.

Baicalein (5,6,7-trihydroxy-2-phenyl-4H-1-benzopyran-4-one), a flavonoid analog from Scutellaria baicalensis, possesses several pharmacological activities including antioxidant, antiproliferative, and anti-inflammatory activities. We previously reported that baicalein inhibits the thymic stromal lymphopoietin (TSLP)/TSLP receptor (TSLPR) signaling pathways and can be used as an active ingredient in the treatment of asthma and atopic dermatitis. However, baicalein is rapidly metabolized to baicalin and baicalein-6-O-glucuronide in vivo, which limits its preclinical and clinical use. In this study, we designed, synthesized, and evaluated baicalein prodrugs that protect the OH group at the 7-position of the A ring in baicalein with the amino acid carbamate functional group. Comprehensive in vitro and in vivo studies identified compound 2 as a baicalein prodrug candidate that improved the plasma exposure of baicalein in mouse animal studies. Our results demonstrated that this prodrug approach could be further adopted to discover oral baicalein prodrugs. Full article

(This article belongs to the Special Issue Anti-inflammatory Effects from Natural Bioactive Compounds—from Bench to Bedside)

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Chemical structure of baicalein and baicalin. Full article ">Figure 2
Stability of baicalein and its carbamate prodrugs in mouse plasma. Test compounds were incubated for 60 min at 37 °C in mouse plasma. % remaining was determined at 60 min vs. time 0 using peak ratios of LC−MS/MS chromatograms. Data are expressed as the mean ± SD (n = 3). Full article ">Figure 3
Metabolic stability of baicalein and its carbamate prodrugs in the mouse liver S9 fractions. Test compounds were incubated at 37 °C with mouse liver S9 fractions in the absence and presence of the cofactor mixture (NADPH, UDPGA, PAPS and GSH). (A) % remaining was determined at 60 min vs. time 0 using peak ratios of LC−MS/MS chromatograms (mean ± SD, n = 3). * p < 0.001 (multiple unpaired t test). (B) Time course of compound 5 disappearance and its conversion to baicalein was determined simultaneously in the mouse liver S9 fractions following incubation of compound 5 in the absence and presence of the cofactor mixture (mean ± SD, n = 3). Full article ">Scheme 1
Synthesis of baicalein carbamate prodrugs. Reagent and conditions: (i) bis-(4-nitrophenyl)carbonate, DIPEA, THF, rt, 12 h; (ii) baicalein, DIPEA, THF, rt, 12 h; (iii) 25% TFA in CH2Cl2, rt, 6 h. Full article ">

20 pages, 5215 KiB

Open AccessArticle

Cellulose Nanofibers Improve the Performance of Retrograded Starch/Pectin Microparticles for Colon-Specific Delivery of 5-ASA

by Andréia Bagliotti Meneguin, Rafael Miguel Sábio, Maurício Palmeira Chaves de Souza, Richard Perosa Fernandes, Anselmo Gomes de Oliveira and Marlus Chorilli

Pharmaceutics 2021, 13(9), 1515; https://doi.org/10.3390/pharmaceutics13091515 - 19 Sep 2021

Cited by 13 | Viewed by 3509

Abstract

Cellulose nanofibers (CNF) were employed as the nanoreinforcement of a retrograded starch/pectin (RS/P) excipient to optimize its colon-specific properties. Although starch retrogradation ranged from 32 to 73%, CNF addition discretely disfavored the RS yield. This result agrees with the finding that in situ [...] Read more.

Cellulose nanofibers (CNF) were employed as the nanoreinforcement of a retrograded starch/pectin (RS/P) excipient to optimize its colon-specific properties. Although starch retrogradation ranged from 32 to 73%, CNF addition discretely disfavored the RS yield. This result agrees with the finding that in situ CNF reduces the presence of the RS crystallinity pattern. A thermal analysis revealed that the contribution of pectin improves the thermal stability of the RS/CNF mixture. Through a complete factorial design, it was possible to optimize the spray-drying conditions to obtain powders with high yield (57%) and low moisture content (1.2%). The powders observed by Field Emission Gum Scanning Electron Microscopy (FEG-SEM) had 1–10 µm and a circular shape. The developed methodology allowed us to obtain 5-aminosalicilic acid-loaded microparticles with high encapsulation efficiency (16–98%) and drug loading (1.97–26.63%). The presence of CNF in RS/P samples was responsible for decreasing the burst effect of release in simulated gastric and duodenal media, allowing the greatest mass of drug to be targeted to the colon. Considering that spray-drying is a scalable process, widely used by the pharmaceutical industry, the results obtained indicate the potential of these microparticles as raw material for obtaining other dosage forms to deliver 5-ASA to the distal parts of gastrointestinal tract, affected by inflammatory bowel disease. Full article

(This article belongs to the Special Issue Polysaccharides-Based Delivery Systems as a Technological Tool to Target Drugs to the Colon)

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RS content (%) as a function of the different proportions of pectin and/or CNF after incubation with an enzymatic solution of pancreatin (0.15 g mL−1). RS quantification was performed through the DNS method for reducing sugar (A). X-ray diffractogram patterns of isolated polymers (P, HAS, RS and CNF) (B), RS in distinct proportions of CNF (RS/CNF1:1, RS/CNF1:0.75, RS/CNF1:0.5 and RS/CNF1:0.25) (C) and RS in the presence of P and distinct proportions of CNF (RS/P/CNF1:1:1, RS/P/CNF1:1:0.75, RS/P/CNF1:1:0.5, RS/P/CNF1:1:0.25) (D). Full article ">Figure 2
TG-DSC curves of isolated compounds and samples retrograded in the presence of pectin and/or CNF. TG curve (A) and DSC curve (B) of RS, HAS, Pectin, CNF. TG curve (C) and DSC curve (D) of RS/CNF and RS/P/CNF in different proportions. DTG curve of the RS in presence of different P and CNF proportions (E). Full article ">Figure 2 Cont.
TG-DSC curves of isolated compounds and samples retrograded in the presence of pectin and/or CNF. TG curve (A) and DSC curve (B) of RS, HAS, Pectin, CNF. TG curve (C) and DSC curve (D) of RS/CNF and RS/P/CNF in different proportions. DTG curve of the RS in presence of different P and CNF proportions (E). Full article ">Figure 3
Pareto chart representing the impact of critical synthesis parameters under residual moisture (A) and process yield (B) and graphic representation of individual factors and their impact on the residual moisture (C) and yield process (D) factors. Full article ">Figure 4
FEG-SEM images of RS/P samples prepared by spray drying employing different values for inlet air temperature, aspirator efficiency and feed flow rate, according to the factorial design outlined in <a href="#pharmaceutics-13-01515-t002" class="html-table">Table 2</a>. The images were obtained with 2000× magnification. Full article ">Figure 5
FEG-SEM images of 5-ASA-loaded RS/P samples prepared by spray drying employing different strategies to improve drug solubility before the drying process: RS/P5ASA0.5% with 500× (a) and 2000× magnification (b), RS/P5ASA1.0% with 500× (c) and 2000× magnification (d), RS/P5ASA-HCl with 500× (e) and 2000× magnification (f), RS/P5ASA-NaOH with 500× (g) and 2000× magnification (h), and RS/P5ASA-polysorbate with 500× (i) and 2000× magnification (j). White arrows indicate the presence of crystals as free drug (unencapsulated). Full article ">Figure 6
FEG/SEM images of 5-ASA-loaded RS/P samples containing different amounts of CNF (10%, 15% and 50%). The images were obtained with 10,000× magnification. Full article ">Figure 7
In vitro 5-ASA release profiles from spray-dried microparticles in media with different pH values, mimicking gastric (0–60 min; pH 1.2), duodenal (60–120 min; pH 4.5) and colonic (120–480 min; pH 6.8) phases. Each value represents the mean ± standard deviation, n = 3. Full article ">

26 pages, 723 KiB

Open AccessArticle

Exploring Drugs and Vaccines Associated with Altered Risks and Severity of COVID-19: A UK Biobank Cohort Study of All ATC Level-4 Drug Categories Reveals Repositioning Opportunities

by Yong Xiang, Kenneth Chi-Yin Wong and Hon-Cheong So

Pharmaceutics 2021, 13(9), 1514; https://doi.org/10.3390/pharmaceutics13091514 - 18 Sep 2021

Cited by 12 | Viewed by 5131

Abstract

Effective therapies for COVID-19 are still lacking, and drug repositioning is a promising approach to address this problem. Here, we adopted a medical informatics approach to repositioning. We leveraged a large prospective cohort, the UK-Biobank (UKBB, N ~ 397,000), and studied associations of [...] Read more.

Effective therapies for COVID-19 are still lacking, and drug repositioning is a promising approach to address this problem. Here, we adopted a medical informatics approach to repositioning. We leveraged a large prospective cohort, the UK-Biobank (UKBB, N ~ 397,000), and studied associations of prior use of all level-4 ATC drug categories (N = 819, including vaccines) with COVID-19 diagnosis and severity. Effects of drugs on the risk of infection, disease severity, and mortality were investigated separately. Logistic regression was conducted, controlling for main confounders. We observed strong and highly consistent protective associations with statins. Many top-listed protective drugs were also cardiovascular medications, such as angiotensin-converting enzyme inhibitors (ACEI), angiotensin receptor blockers (ARB), calcium channel blocker (CCB), and beta-blockers. Some other drugs showing protective associations included biguanides (metformin), estrogens, thyroid hormones, proton pump inhibitors, and testosterone-5-alpha reductase inhibitors, among others. We also observed protective associations by influenza, pneumococcal, and several other vaccines. Subgroup and interaction analyses were also conducted, which revealed differences in protective effects in various subgroups. For example, protective effects of flu/pneumococcal vaccines were weaker in obese individuals, while protection by statins was stronger in cardiovascular patients. To conclude, our analysis revealed many drug repositioning candidates, for example several cardiovascular medications. Further studies are required for validation. Full article

(This article belongs to the Special Issue In Silico Strategies for Prospective Drug Repositionings)

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14 pages, 1927 KiB

Open AccessReview

Functionalized Dendrimer Platforms as a New Forefront Arsenal Targeting SARS-CoV-2: An Opportunity

by Serge Mignani, Xiangyang Shi, Andrii Karpus, Giovanni Lentini and Jean-Pierre Majoral

Pharmaceutics 2021, 13(9), 1513; https://doi.org/10.3390/pharmaceutics13091513 - 18 Sep 2021

Cited by 14 | Viewed by 3825

Abstract

The novel human coronavirus SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) has caused a pandemic. There are currently several marketed vaccines and many in clinical trials targeting SARS-CoV-2. Another strategy is to repurpose approved drugs to decrease the burden of the COVID-19 (official [...] Read more.

The novel human coronavirus SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) has caused a pandemic. There are currently several marketed vaccines and many in clinical trials targeting SARS-CoV-2. Another strategy is to repurpose approved drugs to decrease the burden of the COVID-19 (official name for the coronavirus disease) pandemic. as the FDA (U.S. Food and Drug Administration) approved antiviral drugs and anti-inflammatory drugs to arrest the cytokine storm, inducing the production of pro-inflammatory cytokines. Another view to solve these unprecedented challenges is to analyze the diverse nanotechnological approaches which are able to improve the COVID-19 pandemic. In this original minireview, as promising candidates we analyze the opportunity to develop biocompatible dendrimers as drugs themselves or as nanocarriers against COVID-19 disease. From the standpoint of COVID-19, we suggest developing dendrimers as shields against COVID-19 infection based on their capacity to be incorporated in several environments outside the patients and as important means to stop transmission of SARS-CoV-2. Full article

(This article belongs to the Special Issue Dendrimers and Dendritic Materials against Infectious Diseases)

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22 pages, 7940 KiB

Open AccessArticle

Graphene Oxide and Graphene Quantum Dots as Delivery Systems of Cationic Porphyrins: Photo-Antiproliferative Activity Evaluation towards T24 Human Bladder Cancer Cells

by Luca Menilli, Ana R. Monteiro, Silvia Lazzarotto, Filipe M. P. Morais, Ana T. P. C. Gomes, Nuno M. M. Moura, Sara Fateixa, Maria A. F. Faustino, Maria G. P. M. S. Neves, Tito Trindade and Giorgia Miolo

Pharmaceutics 2021, 13(9), 1512; https://doi.org/10.3390/pharmaceutics13091512 - 18 Sep 2021

Cited by 23 | Viewed by 5061

Abstract

The development of new photodynamic therapy (PDT) agents designed for bladder cancer (BC) treatments is of utmost importance to prevent its recurrence and progression towards more invasive forms. Here, three different porphyrinic photosensitizers (PS) (TMPyP, Zn-TMPyP, and P1-C₅) were non-covalently loaded [...] Read more.

The development of new photodynamic therapy (PDT) agents designed for bladder cancer (BC) treatments is of utmost importance to prevent its recurrence and progression towards more invasive forms. Here, three different porphyrinic photosensitizers (PS) (TMPyP, Zn-TMPyP, and P1-C₅) were non-covalently loaded onto graphene oxide (GO) or graphene quantum dots (GQDs) in a one-step process. The cytotoxic effects of the free PS and of the corresponding hybrids were compared upon blue (BL) and red-light (RL) exposure on T24 human BC cells. In addition, intracellular reactive oxygen species (ROS) and singlet oxygen generation were measured. TMPyP and Zn-TMPyP showed higher efficiency under BL (IC₅₀: 0.42 and 0.22 μm, respectively), while P1-C₅ was more active under RL (IC₅₀: 0.14 μm). In general, these PS could induce apoptotic cell death through lysosomes damage. The in vitro photosensitizing activity of the PS was not compromised after their immobilization onto graphene-based nanomaterials, with Zn-TMPyP@GQDs being the most promising hybrid system under RL (IC₅₀: 0.37 μg/mL). Overall, our data confirm that GO and GQDs may represent valid platforms for PS delivery, without altering their performance for PDT on BC cells. Full article

(This article belongs to the Special Issue Drug Delivery in Photodynamic Therapy (PDT))

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23 pages, 8094 KiB

Open AccessArticle

Multifaced Role of Dual Herbal Principles Loaded-Lipid Nanocarriers in Providing High Therapeutic Efficacity

by Ioana Lacatusu, Teodora Alexandra Iordache, Mirela Mihaila, Dan Eduard Mihaiescu, Anca Lucia Pop and Nicoleta Badea

Pharmaceutics 2021, 13(9), 1511; https://doi.org/10.3390/pharmaceutics13091511 - 18 Sep 2021

Cited by 14 | Viewed by 2747

Abstract

Although many phytochemicals have been used in traditional medicine, there is a great need to refresh the health benefits and adjust the shortcomings of herbal medicine. In this research, two herbal principles (Diosgenin and Glycyrrhiza glabra extract) coopted in the Nanostructured Lipid Carriers [...] Read more.

Although many phytochemicals have been used in traditional medicine, there is a great need to refresh the health benefits and adjust the shortcomings of herbal medicine. In this research, two herbal principles (Diosgenin and Glycyrrhiza glabra extract) coopted in the Nanostructured Lipid Carriers have been developed for improving the most desirable properties of herbal medicine—antioxidant and anti-inflammatory actions. The contribution of phytochemicals, vegetable oils and of lipid matrices has been highlighted by comparative study of size, stability, entrapment efficiency, morphological characteristics, and thermal behavior. According to the in vitro MTS and RTCA results, the dual herbal-NLCs were no cytotoxic toward endothelial cells at concentrations between 25 and 100 µg/mL. A rapid release of Glycyrrhiza glabra and a motivated delay of Diosgenin was detected by the in vitro release experiments. Dual herbal-NLCs showed an elevated ability to annihilate long-life cationic radicals (ABTS^•+) and short-life oxygenated radicals (an inhibition of 63.4% ABTS^•+, while the ability to capture radical oxygen species reached 96%). The production of pro-inflammatory cytokines was significantly inhibited by the newly herbals-NLC (up to 97.9% inhibition of TNF-α and 62.5% for IL-6). The study may open a new pharmacotherapy horizon; it provides a comprehensive basis for the use of herbal-NLC in the treatment of inflammatory diseases. Full article

(This article belongs to the Special Issue Nanotechnology and Natural Products: Plant Bioactive Compounds for Drug Delivery)

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22 pages, 7663 KiB

Open AccessArticle

Co-Injection of Sulfotyrosine Facilitates Retinal Uptake of Hyaluronic Acid Nanospheres Following Intravitreal Injection

by Aiden Eblimit, Mustafa S. Makia, Daniel Strayve, Ryan Crane, Shannon M. Conley, Tirthankar Sinha, Ghanashyam Acharya, Muayyad R. Al-Ubaidi and Muna I. Naash

Pharmaceutics 2021, 13(9), 1510; https://doi.org/10.3390/pharmaceutics13091510 - 18 Sep 2021

Cited by 5 | Viewed by 3075

Abstract

Gene and drug delivery to the retina is a critical therapeutic goal. While the majority of inherited forms of retinal degeneration affect the outer retina, specifically the photoreceptors and retinal pigment epithelium, effective targeted delivery to this region requires invasive subretinal delivery. Our [...] Read more.

Gene and drug delivery to the retina is a critical therapeutic goal. While the majority of inherited forms of retinal degeneration affect the outer retina, specifically the photoreceptors and retinal pigment epithelium, effective targeted delivery to this region requires invasive subretinal delivery. Our goal in this work was to evaluate two innovative approaches for increasing both the persistence of delivered nanospheres and their penetration into the outer retina while using the much less invasive intravitreal delivery method. We formulated novel hyaluronic acid nanospheres (HA-NS, 250 nm and 500 nm in diameter) conjugated to fluorescent reporters and delivered them intravitreally to the adult Balb/C mouse retina. They exhibited persistence in the vitreous and along the inner limiting membrane (ILM) for up to 30 days (longest timepoint examined) but little retinal penetration. We thus evaluated the ability of the small molecule, sulfotyrosine, to disrupt the ILM, and found that 3.2 µg/µL sulfotyrosine led to significant improvement in delivery to the outer retina following intravitreal injections without causing retinal inflammation, degeneration, or loss of function. Co-delivery of sulfotyrosine and HA-NS led to robust improvements in penetration of HA-NS into the retina and accumulation along the interface between the photoreceptors and the retinal pigment epithelium. These exciting findings suggest that sulfotyrosine and HA-NS may be an effective strategy for outer retinal targeting after intravitreal injection. Full article

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37 pages, 1472 KiB

Open AccessPerspective

Inhibition of Scavenger Receptor Class B Type 1 (SR-B1) Expression and Activity as a Potential Novel Target to Disrupt Cholesterol Availability in Castration-Resistant Prostate Cancer

by Mitali Pandey, Grace Cuddihy, Jacob A. Gordon, Michael E. Cox and Kishor M. Wasan

Pharmaceutics 2021, 13(9), 1509; https://doi.org/10.3390/pharmaceutics13091509 - 18 Sep 2021

Cited by 4 | Viewed by 4539

Abstract

There have been several studies that have linked elevated scavenger receptor class b type 1 (SR-B1) expression and activity to the development and progression of castration-resistant prostate cancer (CRPC). SR-B1 facilitates the influx of cholesterol to the cell from lipoproteins in systemic circulation. [...] Read more.

There have been several studies that have linked elevated scavenger receptor class b type 1 (SR-B1) expression and activity to the development and progression of castration-resistant prostate cancer (CRPC). SR-B1 facilitates the influx of cholesterol to the cell from lipoproteins in systemic circulation. This influx of cholesterol may be important for many cellular functions, including the synthesis of androgens. Castration-resistant prostate cancer tumors can synthesize androgens de novo to supplement the loss of exogenous sources often induced by androgen deprivation therapy. Silencing of SR-B1 may impact the ability of prostate cancer cells, particularly those of the castration-resistant state, to maintain the intracellular supply of androgens by removing a supply of cholesterol. SR-B1 expression is elevated in CRPC models and has been linked to poor survival of patients. The overarching belief has been that cholesterol modulation, through either synthesis or uptake inhibition, will impact essential signaling processes, impeding the proliferation of prostate cancer. The reduction in cellular cholesterol availability can impede prostate cancer proliferation through both decreased steroid synthesis and steroid-independent mechanisms, providing a potential therapeutic target for the treatment of prostate cancer. In this article, we discuss and highlight the work on SR-B1 as a potential novel drug target for CRPC management. Full article

(This article belongs to the Collection Pharmaceutical Sciences in Canada)

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Lipoprotein structure—an overview. Lipoproteins are a spherical monolayer of amphipatic phospholipids (yellow) and free/unesterified cholesterol (orange). The tails of the phospholipids create a hydrophobic core of non-polar lipids, primarily cholesterol esters (green) and triglycerides (pink), surrounded by a hydrophilic membrane of phospholipids, free cholesterol and apolipoproteins (blue). Lipoproteins differ in their lipid composition, size, density, major apolipoproteins and function [<a href="#B53-pharmaceutics-13-01509" class="html-bibr">53</a>]. Figure produced using Servier medical art [<a href="#B54-pharmaceutics-13-01509" class="html-bibr">54</a>]. Full article ">Figure 2
Lipoprotein—types and function. Four kinds of lipoproteins exist in the human body viz. high density lipoprotein (HDL), low density lipoprotein (LDL), very low density lipoprotein (VLDL) and one intermediate product, intermediate density lipoprotein. Nomenclature is based on the ratio of their size and density (g/dL): HDL (7–13 nm in diameter, 1.063–1.25), LDL (22–27 nm, 1.019–1.063), ILDL (27–30 nm, 1.006–1.019), VLDL (35–80 nm, 0.95–1.006), chylomicrons (80–1200 nm, <0.95). Their function is determined by the apolipoproteins on their surface. Major apolipoproteins present on lipoproteins include A-I, A-II, C, E in HDL (orange), B-100 in LDL (green); B-100, C, E in LDL (blue), and VLDL (brown) and B-48, A-I, A-II, C, E in chylomicrons (purple). HDL removed cholesterol from peripheral tissue, LDL delivers fat to peripheral tissue, IDL and VLDL transport fats from the liver, while chylomicrons transport dietary fats (food and bile). Figure produced using Servier medical art [<a href="#B54-pharmaceutics-13-01509" class="html-bibr">54</a>]. Full article ">Figure 3
Cholesterol–intracellular and intratumoral metabolism. Cellular uptake of cholesterol is mediated by endocytosis of low density lipoproteins (LDL) via the LDL receptors (LDLRs), and partially trough HDL receptors (HDLRs), ore commonly known as scavenger receptor, Class B1 (SR-B1). LDLR levels in cells is tightly regulated by proprotein convertase subtilisin/kexin type 9 (PCSK9) and inducible degrader of LDLRs (IDOL). In endothelial cells, HDL is a chemotactic factor and signaling is mediated SR-B1 which is associated with (PDZ Domain Containing 1 (PDZK1) and sphingosine phosphate (S1P) receptor 1 (S1PR1) a scaffolding protein, leading to phosphorylation of AKT1 (p-AKT1) which phosphorylates and activates nitric oxide synthase to produce nitric oxide (NO). Cholesterol can also be produced de novo in cells via the mevalonate pathway. Reduction of 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) to mevalonate is controlled by 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMGCR), the enzyme targeted by statin drugs. Cholesterol efflux is facilitated by ATP Binding Cassette Subfamily A Member 1 (ABCA1) transporter. Once in the cell, cholesterol is involved in multiple functions including cell proliferation and androgen synthesis. Excess intracellular cholesterol can get esterified to cholesterol esters by sterol O-acyltransferase 1 (SOAT1) and stored as lipid droplets. Broken and continuous arrows indicate regulation and metabolic steps respectively. Figure produced using Servier medical art [<a href="#B54-pharmaceutics-13-01509" class="html-bibr">54</a>]. Full article ">Figure 4
Role of SR-B1 in the development of foam cells from macrophages. Modified lipoproteins are taken by via SR-B1 present on macrophage. Increased uptake of these modified lipoproteins and cholesterol esters lead to excessive fat in cells, culminating in the formation of foam cells, the main constituents of atherosclerotic lesions. Figure produced using Servier medical art [<a href="#B54-pharmaceutics-13-01509" class="html-bibr">54</a>]. Full article ">

22 pages, 4276 KiB

Open AccessArticle

mPEG-PLGA Nanoparticles Labelled with Loaded or Conjugated Rhodamine-B for Potential Nose-to-Brain Delivery

by Emanuela Fabiola Craparo, Teresa Musumeci, Angela Bonaccorso, Rosalia Pellitteri, Alessia Romeo, Irina Naletova, Lorena Maria Cucci, Gennara Cavallaro and Cristina Satriano

Pharmaceutics 2021, 13(9), 1508; https://doi.org/10.3390/pharmaceutics13091508 - 18 Sep 2021

Cited by 20 | Viewed by 4416

Abstract

Nowdays, neurodegenerative diseases represent a great challenge from both the therapeutic and diagnostic points of view. Indeed, several physiological barriers of the body, including the blood brain barrier (BBB), nasal, dermal, and intestinal barriers, interpose between the development of new drugs and their [...] Read more.

Nowdays, neurodegenerative diseases represent a great challenge from both the therapeutic and diagnostic points of view. Indeed, several physiological barriers of the body, including the blood brain barrier (BBB), nasal, dermal, and intestinal barriers, interpose between the development of new drugs and their effective administration to reach the target organ or target cells at therapeutic concentrations. Currently, the nose-to-brain delivery with nanoformulations specifically designed for intranasal administration is a strategy widely investigated with the goal to reach the brain while bypassing the BBB. To produce nanosystems suitable to study both in vitro and/or in vivo cells trafficking for potential nose-to-brain delivery route, we prepared and characterized two types of fluorescent poly(ethylene glycol)-methyl-ether-block-poly(lactide-co-glycolide) (PLGA–PEG) nanoparticles (PNPs), i.e., Rhodamine B (RhB) dye loaded- and grafted- PNPs, respectively. The latter were produced by blending into the PLGA–PEG matrix a RhB-labeled polyaspartamide/polylactide graft copolymer to ensure a stable fluorescence during the time of analysis. Photon correlation spectroscopy (PCS), UV-visible (UV-vis) spectroscopies, differential scanning calorimetry (DSC), atomic force microscopy (AFM) were used to characterize the RhB-loaded and RhB-grafted PNPs. To assess their potential use for brain targeting, cytotoxicity tests were carried out on olfactory ensheathing cells (OECs) and neuron-like differentiated PC12 cells. Both PNP types showed mean sizes suitable for nose-to-brain delivery (<200 nm, PDI < 0.3) and were not cytotoxic toward OECs in the concentration range tested, while a reduction in the viability on PC12 cells was found when higher concentrations of nanomedicines were used. Both the RhB-labelled NPs are suitable drug carrier models for exploring cellular trafficking in nose-to-brain delivery for short-time or long-term studies. Full article

(This article belongs to the Special Issue Lipid- and/or Polymer-Based Drug Delivery Systems)

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16 pages, 1420 KiB

Open AccessArticle

Modulation of Colorectal Tumor Behavior via lncRNA TP53TG1-Lipidic Nanosystem

by Farimah Masoumi, Sofia M. Saraiva, Belén L. Bouzo, Rafael López-López, Manel Esteller, Ángel Díaz-Lagares and María de la Fuente

Pharmaceutics 2021, 13(9), 1507; https://doi.org/10.3390/pharmaceutics13091507 - 18 Sep 2021

Cited by 5 | Viewed by 3640

Abstract

Long non-coding RNAs (lncRNAs) are an emerging group of RNAs with a crucial role in cancer pathogenesis. In gastrointestinal cancers, TP53 target 1 (TP53TG1) is an epigenetically regulated lncRNA that represents a promising therapeutic target due to its tumor suppressor properties regulating the [...] Read more.

Long non-coding RNAs (lncRNAs) are an emerging group of RNAs with a crucial role in cancer pathogenesis. In gastrointestinal cancers, TP53 target 1 (TP53TG1) is an epigenetically regulated lncRNA that represents a promising therapeutic target due to its tumor suppressor properties regulating the p53-mediated DNA damage and the intracellular localization of the oncogenic YBX1 protein. However, to translate this finding into the clinic as a gene therapy, it is important to develop effective carriers able to deliver exogenous lncRNAs to the targeted cancer cells. Here, we propose the use of biocompatible sphingomyelin nanosystems comprising DOTAP (DSNs) to carry and deliver a plasmid vector encoding for TP53TG1 (pc(TP53TG1)-DSNs) to a colorectal cancer cell line (HCT-116). DSNs presented a high association capacity and convenient physicochemical properties. In addition, pc(TP53TG1)-DSNs showed anti-tumor activities in vitro, specifically a decrease in the proliferation rate, a diminished colony-forming capacity, and hampered migration and invasiveness of the treated cancer cells. Consequently, the proposed strategy displays a high potential as a therapeutic approach for colorectal cancer. Full article

(This article belongs to the Special Issue Non-viral Gene Delivery Systems, 2nd Edition)

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Association of pc(mCherry) to DSNs. (A) Association of pc(mCherry) (1 to 10 µg) to DSNs, prepared with 1% or 10% of DOTAP (DSNs1% and DSNs10%), determined by agarose gel electrophoresis. (B) Physicochemical properties of DSNs1% associating 1 µg of pc(mCherry) and DSNs10% associating 1, 5 and 10 µg of pc(mCherry). (C) Colloidal stability of DSNs10% associating 10 µg of pc(mCherry) in 1% FBS-supplemented DMEM, up to 4 h. (nm, nanometers; PdI, polydispersity index; zeta pot., zeta potential; mV millivolts). Full article ">Figure 2
Nanosystems cytotoxic profile and internalization capacity on HCT-116 cells. (A) Blank nanosystems (SNs, DSNs1%, DSNs10%) and DSNs10% associating 10 µg of the model plasmid encoding mCherry (pc(mCherry)-DSNs10%) were incubated for 24 h with HCT-116 cells up to 5 mg/mL and cell cytotoxicity was determined by MTT. (B) Confocal microscopy images of internalized blank nanosystems labeled with DiR (red channel) and pc(TP53TG1)-DSNs10% (associating 10 µg plasmid) labeled with NBD-SM (green channel). Cell nuclei were counterstained with DAPI (blue channel). Scale bars correspond to 25 µm. (C) FACS analysis of HCT-116 cells positive to pc(TP53TG1)-DSNs labeled with NBD-SM (blue line) upon 4 h of incubation at 37 °C. Ultra-pure water was used as control (red line). Full article ">Figure 3
Transfection efficiency of DSNs in HCT116 cells. (A) Fluorescence microscopy images of mCherry (red signal) expression in cells transfected with a pc(mCherry)-DSNs1% and -DSNs10% for 24 h at 37 °C; (B) Expression of TP53TG1 and its mutated form (MutTP53TG1), in cells transfected with DSNs10% without plasmid (blank) or associating 10 µg of pc(empty), pc(TP53TG1), pc(MutTP53TG1), determined by qRT-PCR using specific primers. The relative expression of each gene was calculated as mean ± SEM of 2^-ddCT normalized to GAPDH. (*** p < 0.001; **** p < 0.0001). Full article ">Figure 4
Tumor suppressor features of pc(empty)-DSNs10%, pc(TP53TG1)-DSNs10% and pc(MutTP53TG1)-DSNs10% in vitro. (A) HCT-116 cells’ viability determined each 24 h up to 72 h, after treatment with DSNs10%, associating different pcDNAs, for 4 h. Data is normalized to time point 0 h. (B) Effect of DSNs10%, associating different pcDNAs on the ability of HCT-116 cells to form colonies. HCT-116 cells were incubated with the nanosystems for 4 h and after 11 days the number of formed colonies was determined. (C) Wound healing assay was performed to evaluate the effect of the nanosystems on HCT-116 cell ability to migrate and close the wound. After creating a wound, the cells were treated with the nanosystems for 4 h (n = 3). The evolution of the wound was monitored up to 96 h through direct observation under light microscope and photos were taken. (D) The migration rate (wound healing assay) was calculated using the acquired photos and data was normalized against time point 0 h. The effects of pc(TP53TG1)-DSNs10% and pc(MutTP53TG1)-DSNs10% were compared to the ones produced by pc(empty)-DSNs10% at each time point. (ns, not significant; * p < 0.05, ** p < 0.01). Full article ">

21 pages, 3745 KiB

Open AccessArticle

Systemically Administered Homing Peptide Targets Dystrophic Lesions and Delivers Transforming Growth Factor-β (TGFβ) Inhibitor to Attenuate Murine Muscular Dystrophy Pathology

by Aqsa Iqbal, Ulrike May, Stuart N. Prince, Tero A.H. Järvinen and Ahlke Heydemann

Pharmaceutics 2021, 13(9), 1506; https://doi.org/10.3390/pharmaceutics13091506 - 18 Sep 2021

Cited by 9 | Viewed by 3666

Abstract

Muscular dystrophy is a progressively worsening and lethal disease, where accumulation of functionality-impairing fibrosis plays a key pathogenic role. Transforming growth factor-β1 (TGFβ1) is a central signaling molecule in the development of fibrosis in muscular dystrophic humans and mice. Inhibition of TGFβ1 has [...] Read more.

Muscular dystrophy is a progressively worsening and lethal disease, where accumulation of functionality-impairing fibrosis plays a key pathogenic role. Transforming growth factor-β1 (TGFβ1) is a central signaling molecule in the development of fibrosis in muscular dystrophic humans and mice. Inhibition of TGFβ1 has proven beneficial in mouse models of muscular dystrophy, but the global strategies of TGFβ1 inhibition produce significant detrimental side effects. Here, we investigated whether murine muscular dystrophy lesion-specific inhibition of TGFβ1 signaling by the targeted delivery of therapeutic decorin (a natural TGFβ inhibitor) by a vascular homing peptide CAR (CARSKNKDC) would reduce skeletal muscle fibrosis and pathology and increase functional characteristics of skeletal muscle. We demonstrate that CAR peptide homes to dystrophic lesions with specificity in two muscular dystrophy models. Recombinant fusion protein consisting of CAR peptide and decorin homes selectively to sites of skeletal muscle damage in mdxDBA2/J and gamma-sarcoglycan deficient DBA2/J mice. This targeted delivery reduced TGFβ1 signaling as demonstrated by reduced nuclear pSMAD staining. Three weeks of targeted decorin treatment decreased both membrane permeability and fibrosis and improved skeletal muscle function in comparison to control treatments in the mdxD2 mice. These results show that selective delivery of decorin to the sites of skeletal muscle damage attenuates the progression of murine muscular dystrophy. Full article

(This article belongs to the Special Issue Precision Delivery of Drugs and Imaging Agents with Peptides)

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15 pages, 1648 KiB

Open AccessArticle

Optimisation of a Microfluidic Method for the Delivery of a Small Peptide

by Felicity Y. Han, Weizhi Xu, Vinod Kumar, Cedric S. Cui, Xaria Li, Xingyu Jiang, Trent M. Woodruff, Andrew K. Whittaker and Maree T. Smith

Pharmaceutics 2021, 13(9), 1505; https://doi.org/10.3390/pharmaceutics13091505 - 18 Sep 2021

Cited by 5 | Viewed by 4231

Abstract

Peptides hold promise as therapeutics, as they have high bioactivity and specificity, good aqueous solubility, and low toxicity. However, they typically suffer from short circulation half-lives in the body. To address this issue, here, we have developed a method for encapsulation of an [...] Read more.

Peptides hold promise as therapeutics, as they have high bioactivity and specificity, good aqueous solubility, and low toxicity. However, they typically suffer from short circulation half-lives in the body. To address this issue, here, we have developed a method for encapsulation of an innate-immune targeted hexapeptide into nanoparticles using safe non-toxic FDA-approved materials. Peptide-loaded nanoparticles were formulated using a two-stage microfluidic chip. Microfluidic-related factors (i.e., flow rate, organic solvent, theoretical drug loading, PLGA type, and concentration) that may potentially influence the nanoparticle properties were systematically investigated using dynamic light scattering and transmission electron microscopy. The pharmacokinetic (PK) profile and biodistribution of the optimised nanoparticles were assessed in mice. Peptide-loaded lipid shell-PLGA core nanoparticles with designated size (~400 nm) and a sustained in vitro release profile were further characterized in vivo. In the form of nanoparticles, the elimination half-life of the encapsulated peptide was extended significantly compared with the peptide alone and resulted in a much higher distribution into the lung. These novel nanoparticles with lipid shells have considerable potential for increasing the circulation half-life and improving the biodistribution of therapeutic peptides to improve their clinical utility, including peptides aimed at treating lung-related diseases. Full article

(This article belongs to the Special Issue Nanomedicine Formulations Based on PLGA Nanoparticles for Diagnosis, Monitoring and Treatment of Disease: From Bench to Bedside)

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Schematic illustration of the microfluidic chip and the nanoparticle formulation process. There are four inlets and one outlet in the microfluidic chip. Peptide-PLGA solution is injected through the middle inlet while water is injected through two side inlets. The peptide-loaded PLGA nanoparticles were formed at this stage. Lipid injected through the central inlet forms the lipid shell structure of the PLGA nanoparticles. Fabricated peptide-loaded lipid shell-polymer core nanoparticles are collected from the outlet. Full article ">Figure 2
Mean (±SEM) (n = 3) size (a) and PDI (b) of ‘blank’ PLGA nanoparticles formulated using 1% PLGA in DCM, ACN, and TFE + DMF (3:7, v/v) under a range of side/centre flow rate conditions (water inside inlets to solvent in centre inlet, 60/4.5 = 60 to 4.5 mL/h, 120/3 = 120 to 3 mL/h, 120/1 = 120 to 1 mL/h). Using the same solvent, increasing flow rate and flow rate ratios significantly decreased the size of nanoparticles. Increasing flow rate or flow rate ratio caused a dramatic decrease in the PDI of nanoparticles formulated using DCM but did not significantly affect nanoparticles formulated using ACN or TFE + DMF (3:7). Two-way ANOVA analysis followed by Tukey test for multiple comparisons (**** p < 0.0001; ** p < 0.01; * p < 0.05; and ns, no significant difference). Full article ">Figure 3
Mean (±SEM) (n = 3) size (a) and PDI (b) of blank nanoparticles formulated using a low sides/centre flow rate ratio (60/4.5) using 1% PLGA in a range of organic solvents (DCM, EA, TFE + DMF (3:7, v/v), ACN, and acetone). Nanoparticles fabricated using DCM had the largest size (400 nm) and the highest PDI (0.8). By comparison, use of ACN resulted in particles of the smallest size (<200 nm) and lowest PDI (~0.2). Mean (±SEM) (n = 3) size (c) and PDI (d) of blank nanoparticles formulated using a high sides/centre flow rate ratio (120/3) using 1% PLGA in different organic solvents (DCM, EA, TFE + DMF (3:7, v/v), and ACN). Both DCM and EA produced particles of a larger size (~250 nm) compared with ACN (~100 nm). DCM provided the highest PDI (0.6) while ACN led to the lowest PDI (<0.2). Mean (±SEM) (n = 3) size (e) and PDI (f) of 5% theoretical drug-loaded nanoparticles formulated with a low side/centre flow rate ratio (60/4.5) using 1% PLGA in different organic solvents (DCM, EA, and ACN). DCM provided a larger particle size (>400 nm) compared with EA (~350 nm) and ACN (150 nm). Solvents did not show a significant difference in PDI (0.2~0.3). One-way ANOVA analysis followed by Tukey test for multiple comparisons (**** p < 0.0001; *** p < 0.001; ** p < 0.01; * p < 0.05; and ns, no significant difference). Full article ">Figure 4
Mean (±SEM) (n = 3) size (a) and PDI (b) of nanoparticles with a range of theoretical drug loadings (0, 5, 10, and 20%) fabricated using 1% PLGA in DCM using a low side/centre flow rate ratio (60/4.5). The mean size of nanoparticles increased with increased drug loading, with this effect most marked at 20% peptide loading (0 < 5 < 10 < 20%). The PDI was significantly decreased for particles containing 5% peptide (<0.4) compared with blank nanoparticles (0.8). PDI increased with increased drug loading (5 < 10 < 20%). One-way ANOVA analysis followed by Tukey test for multiple comparisons (** p < 0.01; and ns, no significant difference). Full article ">Figure 5
Mean (±SEM) (n = 3) size (a), PDI (b), and Z-potential (c) of peptide-loaded nanoparticles (5% theoretical drug loading) fabricated using 1% PLGA in DCM or 1% PLGA + 1% PEG-PLGA in DCM under low side/centre flow rate ratio conditions (60/4.5). The extra 1% PEG-PLGA showed a slight increment in size distribution but significantly lower Z-potential. One-way ANOVA analysis followed by Tukey test for multiple comparisons (**** p < 0.0001; and ns, no significant difference). Full article ">Figure 6
(a) Mean (±SEM) in vitro C5aR1 peptide antagonist release profiles (n = 3) for nanoparticles formulated using 1% PLGA in DCM or 1% PLGA + 1% PEG-PLGA in DCM using a low side/centre flow rate ratio (60/4.5). The percentage drug release was calculated based on actual drug loading. Two-way ANOVA analysis followed by Sidak’s test for multiple comparisons (**** p < 0.0001; and ** p < 0.01). Nanoparticles prepared using 1% PLGA + 1%PEG-PLGA in DCM were characterized for morphology (b) using transmission electron microscopy (TEM) and size distribution (intensity%) (c) using DLS. The TEM image indicates that peptide nanoparticles were successfully coated by a lipid shell as the foggy boundary could be observed. The shadowy particles in the background are components that did not coat polymer particles. The scale bar is 400 nm. Full article ">Figure 7
(a) Plasma concentration versus time profile (two-compartmental model) (mean ± SEM, n = 4) of the C5aR1 peptide antagonist administered by bolus intravenous (IV) injection at a dose of 1 mg/kg either as free compound (black) or in the form of lipid-shell PLGA nanoparticles (pink). (b) Biodistribution (mean ± SEM, n = 4) of peptide released from nanoparticles in different organs (brain, spinal cord, liver, lung, kidney, spleen) (ng/g). Tissues were extracted on day one and day five after a single IV injection of 1 mg/kg either as free peptide (black on day one due to the short half-life of this peptide based on our previous PK studies for the free peptide) or in the form of nanoparticles (pink on day five), respectively. Statistical analysis was conducted by two-way ANOVA, followed by Sidak’s test for multiple comparisons (**** p < 0.0001). Full article ">

18 pages, 4916 KiB

Open AccessArticle

A Recombinant Fusion Construct between Human Serum Albumin and NTPDase CD39 Allows Anti-Inflammatory and Anti-Thrombotic Coating of Medical Devices

by Meike-Kristin Abraham, Elena Jost, Jan David Hohmann, Amy Kate Searle, Viktoria Bongcaron, Yuyang Song, Hans Peter Wendel, Karlheinz Peter, Stefanie Krajewski and Xiaowei Wang

Pharmaceutics 2021, 13(9), 1504; https://doi.org/10.3390/pharmaceutics13091504 - 18 Sep 2021

Cited by 6 | Viewed by 3337

Abstract

Medical devices directly exposed to blood are commonly used to treat cardiovascular diseases. However, these devices are associated with inflammatory reactions leading to delayed healing, rejection of foreign material or device-associated thrombus formation. We developed a novel recombinant fusion protein as a new [...] Read more.

Medical devices directly exposed to blood are commonly used to treat cardiovascular diseases. However, these devices are associated with inflammatory reactions leading to delayed healing, rejection of foreign material or device-associated thrombus formation. We developed a novel recombinant fusion protein as a new biocompatible coating strategy for medical devices with direct blood contact. We genetically fused human serum albumin (HSA) with ectonucleoside triphosphate diphosphohydrolase-1 (CD39), a promising anti-thrombotic and anti-inflammatory drug candidate. The HSA-CD39 fusion protein is highly functional in degrading ATP and ADP, major pro-inflammatory reagents and platelet agonists. Their enzymatic properties result in the generation of AMP, which is further degraded by CD73 to adenosine, an anti-inflammatory and anti-platelet reagent. HSA-CD39 is functional after lyophilisation, coating and storage of coated materials for up to 8 weeks. HSA-CD39 coating shows promising and stable functionality even after sterilisation and does not hinder endothelialisation of primary human endothelial cells. It shows a high level of haemocompatibility and diminished blood cell adhesion when coated on nitinol stents or polyvinylchloride tubes. In conclusion, we developed a new recombinant fusion protein combining HSA and CD39, and demonstrated that it has potential to reduce thrombotic and inflammatory complications often associated with medical devices directly exposed to blood. Full article

(This article belongs to the Special Issue Albumin-Based Drug Delivery Systems)

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21 pages, 5447 KiB

Open AccessArticle

Hyaluronic Acid-Binding, Anionic, Nanoparticles Inhibit ECM Degradation and Restore Compressive Stiffness in Aggrecan-Depleted Articular Cartilage Explants

by Marcus Deloney, Parssa Garoosi, Vanessa F. C. Dartora, Blaine A. Christiansen and Alyssa Panitch

Pharmaceutics 2021, 13(9), 1503; https://doi.org/10.3390/pharmaceutics13091503 - 18 Sep 2021

Cited by 6 | Viewed by 3154

Abstract

Joint trauma results in the production of inflammatory cytokines that stimulate the secretion of catabolic enzymes, which degrade articular cartilage. Molecular fragments of the degraded articular cartilage further stimulate inflammatory cytokine production, with this process eventually resulting in post-traumatic osteoarthritis (PTOA). The loss [...] Read more.

Joint trauma results in the production of inflammatory cytokines that stimulate the secretion of catabolic enzymes, which degrade articular cartilage. Molecular fragments of the degraded articular cartilage further stimulate inflammatory cytokine production, with this process eventually resulting in post-traumatic osteoarthritis (PTOA). The loss of matrix component aggrecan occurs early in the progression of PTOA and results in the loss of compressive stiffness in articular cartilage. Aggrecan is highly sulfated, associates with hyaluronic acid (HA), and supports the compressive stiffness in cartilage. Presented here, we conjugated the HA-binding peptide GAHWQFNALTVRGSG (GAH) to anionic nanoparticles (hNPs). Nanoparticles conjugated with roughly 19 GAH peptides, termed 19 GAH-hNP, bound to HA in solution and increased the dynamic viscosity by 94.1% compared to an HA solution treated with unconjugated hNPs. Moreover, treating aggrecan-depleted (AD) cartilage explants with 0.10 mg of 19 GAH-hNP restored the cartilage compressive stiffness to healthy levels six days after a single nanoparticle treatment. Treatment of AD cartilage with 0.10 mg of 19 GAH-hNP inhibited the degradation of articular cartilage. Treated AD cartilage had 409% more collagen type II and 598% more GAG content than untreated-AD explants. The 19 GAH-hNP therapeutic slowed ECM degradation in AD cartilage explants, restored the compressive stiffness of damaged cartilage, and showed promise as a localized treatment for PTOA. Full article

(This article belongs to the Special Issue Polymer Therapeutics: From Synthesis to Biomedical Applications)

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21 pages, 4178 KiB

Open AccessArticle

Comparative In Vitro Evaluation of Commercial Periodontal Gels on Antibacterial, Biocompatibility and Wound Healing Ability

by Marta Munar-Bestard, Maria Antonia Llopis-Grimalt, Joana Maria Ramis and Marta Monjo

Pharmaceutics 2021, 13(9), 1502; https://doi.org/10.3390/pharmaceutics13091502 - 18 Sep 2021

Cited by 9 | Viewed by 4273

Abstract

In the last years, several studies testing commercial periodontal gels that contain chlorhexidine (CHX) or other antibacterial agents, have raised concerns regarding their cytotoxicity in periodontal tissues. We aimed at comparing the biocompatibility but also the efficacy as regards to the antibacterial and [...] Read more.

In the last years, several studies testing commercial periodontal gels that contain chlorhexidine (CHX) or other antibacterial agents, have raised concerns regarding their cytotoxicity in periodontal tissues. We aimed at comparing the biocompatibility but also the efficacy as regards to the antibacterial and wound healing ability of different commercial periodontal gels. In vitro human gingival fibroblasts (GF) and a 3D model of human tissue equivalents of gingiva (GTE) were used under inflammatory conditions to evaluate wound closure, cytotoxicity and gene expression. Antibacterial effects were also investigated on Porphyromonas gingivalis growth, viability and gingipain activity. In GF and in the bacterial study, we found cytotoxic effects on GF and a high inhibition on bacterial growth rate in gels containing CHX, asiaticoside, enoxolone, cetylpyridinium chloride, propolis and eugenol. Of the two gels that were non-cytotoxic, Syntoss Biogel (containing chondrontin sulfate) and Emdogain (EMD, containing amelogenin and propylene glycol alginate), EMD showed the best wound closure, with no effect on P. gingivalis growth but decreased gingipain activity. On the other hand, Syntoss Biogel reduced viability and gingipain activity of P. gingivalis, but lack wound healing capacity. In the 3D GTE, Syntoss Biogel and EMD showed a good biocompatibility. Among all the tested gels, formulations containing CHX, asiaticoside, enoxolone, cetylpyridinium chloride, propolis and eugenol showed high antibacterial effect but also showed high cytotoxicity in eukaryotic cells. EMD was the one with the best biocompatibility and wound healing ability at the conditions tested. Full article

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23 pages, 8062 KiB

Open AccessArticle

Surface-Modified Multifunctional Thymol-Loaded Biodegradable Nanoparticles for Topical Acne Treatment

by Camila Folle, Natalia Díaz-Garrido, Elena Sánchez-López, Ana Maria Marqués, Josefa Badia, Laura Baldomà, Marta Espina, Ana Cristina Calpena and María Luisa García

Pharmaceutics 2021, 13(9), 1501; https://doi.org/10.3390/pharmaceutics13091501 - 18 Sep 2021

Cited by 19 | Viewed by 3955

Abstract

The present work is focused on the development of novel surface-functionalized poly(lactic-co-glycolic acid) nanoparticles loaded with thymol (TH-NPs) for topical administration enhancing thymol anti-inflammatory, antioxidant and wound healing activities against acne. TH-NPs were prepared by solvent evaporation method using different surface functionalization strategies [...] Read more.

The present work is focused on the development of novel surface-functionalized poly(lactic-co-glycolic acid) nanoparticles loaded with thymol (TH-NPs) for topical administration enhancing thymol anti-inflammatory, antioxidant and wound healing activities against acne. TH-NPs were prepared by solvent evaporation method using different surface functionalization strategies and obtaining suitable physicochemical parameters and a good short-term stability at 4 °C. Moreover, TH-NPs skin penetration and antioxidant activity were assessed in ex vivo pig skin models. Skin penetration of TH-NPs followed the follicular route, independently of the surface charge and they were able to enhance antioxidant capacity. Furthermore, antimicrobial activity against Cutibacterium acnes was evaluated in vitro by the suspension test showing improved antibacterial performance. Using human keratinocyte cells (HaCat), cytotoxicity, cellular uptake, antioxidant, anti-inflammatory and wound healing activities were studied. TH-NPs were non-toxic and efficiently internalized inside the cells. In addition, TH-NPs displayed significant anti-inflammatory, antioxidant and wound healing activities, which were highly influenced by TH-NPs surface modifications. Moreover, a synergic activity between TH-NPs and their surface functionalization was demonstrated. To conclude, surface-modified TH-NPs had proven to be suitable to be used as anti-inflammatory, antioxidant and wound healing agents, constituting a promising therapy for treating acne infection and associated inflammation. Full article

(This article belongs to the Special Issue Controlled Release of Nanostructured Drug Systems)

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28 pages, 2838 KiB

Open AccessReview

Iterative Upgrading of Small Molecular Tyrosine Kinase Inhibitors for EGFR Mutation in NSCLC: Necessity and Perspective

by Jing Zhu, Qian Yang and Weiguo Xu

Pharmaceutics 2021, 13(9), 1500; https://doi.org/10.3390/pharmaceutics13091500 - 18 Sep 2021

Cited by 12 | Viewed by 4343

Abstract

Molecular targeted therapy has been reported to have fewer adverse effects, and offer a more convenient route of administration, compared with conventional chemotherapy. With the development of sequencing technology, and research on the molecular biology of lung cancer, especially whole-genome information on non-small [...] Read more.

Molecular targeted therapy has been reported to have fewer adverse effects, and offer a more convenient route of administration, compared with conventional chemotherapy. With the development of sequencing technology, and research on the molecular biology of lung cancer, especially whole-genome information on non-small cell lung cancer (NSCLC), various therapeutic targets have been unveiled. Among the NSCLC-driving gene mutations, epidermal growth factor receptor (EGFR) mutations are the most common, and approximately 10% of Caucasian, and more than 50% of Asian, NSCLC patients have been found to have sensitive EGFR mutations. A variety of targeted therapeutic agents for EGFR mutations have been approved for clinical applications, or are undergoing clinical trials around the world. This review focuses on: the indications of approved small molecular kinase inhibitors for EGFR mutation-positive NSCLC; the mechanisms of drug resistance and the corresponding therapeutic strategies; the principles of reasonable and precision molecular structure; and the drug development discoveries of next-generation inhibitors for EGFR. Full article

(This article belongs to the Special Issue Protein Kinase Inhibitors for Targeted Anticancer Therapies)

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The timeline of EGFR-TKI discovery for targeted therapy of NSCLC in the U. S. and in China, respectively. EGFR-TKIs: epidermal growth factor receptor kinase inhibitors; ALK: anaplastic lymphoma kinase. Full article ">Figure 2
Mechanism and mutation frequency of acquired resistance to EGFR-TKIs. Reprinted with permission from ref. [<a href="#B52-pharmaceutics-13-01500" class="html-bibr">52</a>]. Copyright 2018 Springer Nature. Full article ">Figure 3
The acquired resistance mechanisms to third-generation EGFR-TKIs can be classified as EGFR-dependent resistance and EGFR-independent resistance. The EGFR-dependent mechanism: EGFR re mutation, T790M deletion and EGFR amplification; The EGFR-independent mechanism: amplification of MET and HER2, mutation of RAS and BRAF, histological transformation, EMT, oncogene fusion, activation of the PI3K/Akt/mTOR pathway, PTEN deletion, abnormity of the FGFR signaling pathway, and the induction of angiogenesis. (MET: mitogen activation kinase; EMT: epithelial mesenchymal transition; PTEN: phosphatase and tensin homology; FGFR: fibroblast growth factor receptor; ERK: extracellular regulated protein kinases). Reprinted with permission from ref. [<a href="#B74-pharmaceutics-13-01500" class="html-bibr">74</a>]. Copyright 2018 Elsevier Science. Full article ">Figure 4
Difference of acquired resistance mechanisms between first-line (A) and second-line (B) treatment of third-generation TKI osimertinib. Adapted from [<a href="#B76-pharmaceutics-13-01500" class="html-bibr">76</a>,<a href="#B77-pharmaceutics-13-01500" class="html-bibr">77</a>], published by Future Medicine, 2018 and Elsevier, 2018. Full article ">Figure 5
Differences in drug resistance mechanisms between first-line and second-line after the third-generation EGFR-TKIs treatment. Full article ">Figure 6
Comprehensive post-therapeutic strategies of drug resistance for EGFR mutant NSCLC-targeted therapy. The colored ball represents acquired resistance mechanism, which indicates the heterogeneity of tumor (JAK: Janus-activated kinase). Reprinted with permission from ref. [<a href="#B115-pharmaceutics-13-01500" class="html-bibr">115</a>]. Copyright 2018 ACS American Chemical Society. Full article ">

24 pages, 6962 KiB

Open AccessArticle

Indocyanine Green-Camptothecin Co-Loaded Perfluorocarbon Double-Layer Nanocomposite: A Versatile Nanotheranostics for Photochemotherapy and FDOT Diagnosis of Breast Cancer

by Yu-Hsiang Lee, Po-Wei Kuo, Chun-Ju Chen, Chu-Jih Sue, Ya-Fen Hsu and Min-Chun Pan

Pharmaceutics 2021, 13(9), 1499; https://doi.org/10.3390/pharmaceutics13091499 - 17 Sep 2021

Cited by 5 | Viewed by 2582

Abstract

Breast cancer remains the most frequently diagnosed cancer and is the leading cause of neoplastic disease burden for females worldwide, suggesting that effective therapeutic and/or diagnostic strategies are still urgently needed. In this study, a type of indocyanine green (ICG) and camptothecin (CPT) [...] Read more.

Breast cancer remains the most frequently diagnosed cancer and is the leading cause of neoplastic disease burden for females worldwide, suggesting that effective therapeutic and/or diagnostic strategies are still urgently needed. In this study, a type of indocyanine green (ICG) and camptothecin (CPT) co-loaded perfluorocarbon double-layer nanocomposite named ICPNC was developed for detection and photochemotherapy of breast cancer. The ICPNCs were designed to be surface modifiable for on-demand cell targeting and can serve as contrast agents for fluorescence diffuse optical tomography (FDOT). Upon near infrared (NIR) irradiation, the ICPNCs can generate a significantly increased production of singlet oxygen compared to free ICG, and offer a comparable cytotoxicity with reduced chemo-drug dosage. Based on the results of animal study, we further demonstrated that the ICPNCs ([ICG]/[CPT] = 40-/7.5-μM) in association with 1-min NIR irradiation (808 nm, 6 W/cm²) can provide an exceptional anticancer effect to the MDA-MB-231 tumor-bearing mice whereby the tumor size was significantly reduced by 80% with neither organ damage nor systemic toxicity after a 21-day treatment. Given a number of aforementioned merits, we anticipate that the developed ICPNC is a versatile theranostic nanoagent which is highly promising to be used in the clinic. Full article

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Full article ">Figure 1
Assessment of physicochemical properties of the ICPNCs and HICPNCs. (a) The ICPNCs were synthesized through double emulsification processes using polyethoxylated fluorosurfactant and CT-F68 as the emulsifiers. The HICPNCs were obtained after conjugation with anti-HER2-mAb on the ICPNC surface. (b) Photograph of the phantom setup using 1S-1D scanning platform. The schematic drawings I and II display the relative positions of the BTiM, BTuM, laser light source, and light detector from side (I) and top (II) views, respectively. (c) SEM image of the ICPNCs at 15,000× magnification. The inset photographs exhibit the TEM image (20,000×) of the ICPNC (I) and the real appearance of the ICPNC sample before (II) and after (III) centrifugation. (d) Size and surface charge (ζ—potential) distribution profiles of the ICPNCs and HICPNCs measured by DLS. Values are mean ± s.d. (n = 3). (e) Verification of the presence and bioactivity of anti-HER2-mAb on the ICPNC surface. The intensity of fluorescence expressed from each group was measured by spectrofluorometry performed with 488/525 nm of excitation/emission wavelength and was quantitatively represented by RFUs. Values are mean ± s.d. (n = 3). * p < 0.05. Full article ">Figure 2
Assessment of stability and functionality of the ICPNCs in vitro. (a) Quantitative analysis of ICG remaining in the ICPNCs or PBS under incubation at 4 or 37 °C for 48 h. (b) Cumulative release profiles of the ICPNC-encapsulated CPT under incubation at 4 or 37 °C in PBS for 48 h. (c,d) Temperature elevation profiles of freely dissolved ICG (c) and the ICPNCs (d) under NIR irradiation for 5 min. (e,f) Singlet oxygen productions of freely dissolved ICG (e) and the ICPNCs (f) under NIR irradiation for 5 min. The yield of singlet oxygen was assessed based on the intensity of the SOSG-induced fluorescence measured by spectrofluorometry performed with 488/525 nm of excitation/emission wavelength and quantitatively represented by RFUs. In (c–f), groups with different ICG concentrations are denoted by different line colors as indicated in the figure. NIR irradiation was performed using an 808-nm laser with an output intensity of 6 W/cm2 for 5 min. (g) Verification of HER2-binding specificity of the HICPNCs. The histogram shows the fluorescence levels of the BT-474 cells after treatment with ICPNCs or HICPNCs in the presence (competitive; 1 or 2 μg/mL) and absence (non-competitive) of free anti-HER2-mAb for 4 h. The ICPNCs and HICPNCs containing 40 µM ICG and 7.5 µM CPT were employed for the experiment. The intensities of ICG-derived fluorescence were measured by spectrofluorometry performed with 750/838 nm of excitation/emission wavelength and were quantitatively represented by RFUs. Values in (a–g) are mean ± s.d. (n = 3). * p < 0.05. Full article ">Figure 3
Cytotoxicity of the ICPNCs to breast cancer cells in vitro. (a) Photomicrographic images of MDA-MB-453 cells under various treatments. Rows A–D represent the groups treated with CPT, free ICG + NIR, ICPNCs, and ICPNCs + NIR, respectively, in which the concentrations of ICG and/or CPT (ICG/CPT) were set as 2/0.375, 4/0.75, 20/3.75, 40/7.5, and 80/15 μM as indicated in the figure. NIR irradiation was performed using an 808-nm laser with an output intensity of 6 W/cm2 for 5 min. ×1 denotes the cells with neither drug nor NIR exposure. ×2 represents the cells treated with NIR exposure for 5 min followed by incubation at 37 °C for 24 h. The green and red cells stained by calcein-AM and propidium iodide represent live and dead cells, respectively. All images were photographed using a fluorescence microscope at 200X magnification. Scale bar = 30 μm. (b,c) Quantitative analyses of the viabilities of MDA-MB-453 (b) and BT-474 (c) cells after treatment with different conditions as indicated on the X-axis. Values are mean ± s.d. (n = 3). * p < 0.05 compared to the group without treatment. ** p < 0.05 compared to the group with NIR exposure alone. *** p < 0.05 compared to the group with equal concentration of free CPT. **** p < 0.05 compared to the group with free ICG + NIR under equal concentration setting. ***** p < 0.05 compared to the group with equal dose of ICPNCs without NIR irradiation. Full article ">Figure 4
Analyses of FDOT fluorescence signals using free ICG or ICPNCs as the contrast agent in phantom study. Rows A–D represent the groups using free ICG with RI = 0%, 30%, 70%, and 100%, while E–G denote the groups using ICPNCs containing 1, 5, and 10 μM of ICG with RI = 100% as indicated in the figure. Columns 1–4 denote the positions of the source light as illustrated in <a href="#pharmaceutics-13-01499-f001" class="html-fig">Figure 1</a>(bII). The fluorescence intensities expressed from various positions were analyzed using both simulated and algorithmic approaches. The FDOT was performed using an NIR laser with an intensity of 30 mW and the integration time was set as 800 ms. * NIRCA denotes NIR contrast agent. † FL represents fluorescence. Full article ">Figure 5
Image reconstructions of BTiM and BTuM using free ICG or ICPNCs as the NIR contrast agent. (A–J) represent the reconstructed images gained by FDOT or DOT with various conditions as indicated in the figure. The blue circle denotes the true position of the BTuM in each image determined based on the known experimental settings. Full article ">Figure 6
Tumoricidal effect of the ICPNCs in vivo. (a) Time-lapse NIR fluorescent images of tumor-bearing mice after intratumoral injection of free ICG (100 μM; A1–M1) or ICPNCs ([ICG] = 100 μM; A2–M2). The black circle denotes the real position of the tumor in vivo. (b) Variations of ICG-derived fluorescence expressed from the xenograft MDA-MB-231 tumors injected with free ICG or ICPNCs within 60 min. The intensity of the fluorescence was quantitatively represented by RFUs. Values are mean ± s.d. (n = 3). (c) Appearances of the MDA-MB-231 tumor-bearing nude mice with treatment of PBS, free CPT, IPNCs + NIR, ICPNCs, or ICPNCs + NIR as indicated in the figure. The condition of the tumor in each mouse was photographed every 72 h for 21 days before treatment or sacrifice. NIR irradiation was performed using an 808 nm laser with an output intensity of 6 W/m2 for 60 s. The concentrations of ICG and CPT in the settings of IPNCs and/or free CPT were equal to the dosages provided by the ICPNCs set as [ICG]/[CPT] = 40/7.5-μM. (d) Photographs of the tumors harvested after the mice were sacrificed on the 21st day. (e–g) Variations in tumor size (e), body weight (f), and survival rate (g) of all the experimental mice within 21-day treatment. Values in (e,f) are mean ± s.d. (n = 5). The survival rate (g) of each group was calculated based on the total live/dead numbers of mice employed for obtaining five live mice on the 21st day. Full article ">Figure 7
Histological analysis of skin tissue in the vicinity of the tumor. (a,b) Photomicrographic images of H&E-stained (a) or IL-1b IHC-stained (b) skin tissues at 4× (A1–E1) and 20× (A2–E2) magnifications. Columns A–E denote different treatments as indicated in the figure. Black arrows in A1–E1 indicate the tumor in the image. The photographs A2–E2 are the magnified images of the area marked by the dashed block in A1–E1. Full article ">Figure 8
Blood biochemical analyses of the experimental mice. The expression levels of liver (a,b) and kidney (c,d) functional markers as well as the numbers of white blood cells (e), red blood cells (f), and platelets (g) of all the experimental mice were measured 24 h before treatment (day −1) and at the moment before sacrifice (day 21). Values are the mean ± s.d. (n = 5). Full article ">Figure 9
In vivo toxicological study of the ICPNCs. (a) Quantitative analyses of the amounts of CPT remaining in the heart, liver, spleen, lung, kidney, and tumor of the MDA-MB-231 tumor-bearing nude mice after treatment with PBS, CPT, ICPNCs, or ICPNCs + NIR for 21 days. Values are the mean ± s.d. (n = 5). * p < 0.05 compared to the value obtained from the group with PBS for the same organ. † p < 0.05. (b) Photomicrographic images of H&E-stained heart, liver, spleen, lung, and kidney obtained from the MDA-MB-231 tumor-bearing nude mice with various treatments for 21 days as indicated in the figure. Full article ">

16 pages, 1503 KiB

Open AccessArticle

Investigating the Central Nervous System Disposition of Actinomycin D: Implementation and Evaluation of Cerebral Microdialysis and Brain Tissue Measurements Supported by UPLC-MS/MS Quantification

by Julia Benzel, Gzona Bajraktari-Sylejmani, Philipp Uhl, Abigail Davis, Sreenath Nair, Stefan M. Pfister, Walter E. Haefeli, Johanna Weiss, Jürgen Burhenne, Kristian W. Pajtler and Max Sauter

Pharmaceutics 2021, 13(9), 1498; https://doi.org/10.3390/pharmaceutics13091498 - 17 Sep 2021

Cited by 3 | Viewed by 4302

Abstract

Actinomycin D is a potent cytotoxic drug against pediatric (and other) tumors that is thought to barely cross the blood–brain barrier. To evaluate its potential applicability for the treatment of patients with central nervous system (CNS) tumors, we established a cerebral microdialysis model [...] Read more.

Actinomycin D is a potent cytotoxic drug against pediatric (and other) tumors that is thought to barely cross the blood–brain barrier. To evaluate its potential applicability for the treatment of patients with central nervous system (CNS) tumors, we established a cerebral microdialysis model in freely moving mice and investigated its CNS disposition by quantifying actinomycin D in cerebral microdialysate, brain tissue homogenate, and plasma. For this purpose, we developed and validated an ultraperformance liquid chromatography–tandem mass spectrometry assay suitable for ultra-sensitive quantification of actinomycin D in the pertinent biological matrices in micro-samples of only 20 µL, with a lower limit of quantification of 0.05 ng/mL. In parallel, we confirmed actinomycin D as a substrate of P-glycoprotein (P-gp) in in vitro experiments. Two hours after intravenous administration of 0.5 mg/kg, actinomycin D reached total brain tissue concentrations of 4.1 ± 0.7 ng/g corresponding to a brain-to-plasma ratio of 0.18 ± 0.03, while it was not detectable in intracerebral microdialysate. This tissue concentration exceeds the concentrations of actinomycin D that have been shown to be effective in in vitro experiments. Elimination of the drug from brain tissue was substantially slower than from plasma, as shown in a brain-to-plasma ratio of approximately 0.53 after 22 h. Because actinomycin D reached potentially effective concentrations in brain tissue in our experiments, the drug should be further investigated as a therapeutic agent in potentially susceptible CNS malignancies, such as ependymoma. Full article

(This article belongs to the Special Issue Quantification of Therapeutic Peptides by LC-MS)

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Positive product spectrum (MS/MS) of the [M+H]+ signal (m/z 1255.6) of actinomycin D in collision-induced decomposition using a collision energy of 62 V. The structures of actinomycin D, actinomycin C, and their monitored common product ion (m/z 459.1) are additionally depicted. For details on the product ions and fragmentation scheme of actinomycin D, the reader is referred to the work of Thomas and co-workers [<a href="#B29-pharmaceutics-13-01498" class="html-bibr">29</a>]. Full article ">Figure 2
Selected UPLC-MS/MS chromatograms of the processed brain tissue homogenate samples, with the analyte transition in black and internal standard (IS) transition in grey: (A) blank sample, (B) sample with added IS, (C) sample at a lower limit of quantification (LLOQ) level (representing 0.050 ng/mL), (D) sample at a mid QC concentration (representing 3.00 ng/mL), and (E) brain tissue sample 2 h after intravenous administration of 0.5 mg/kg actinomycin D to mouse #1 (calculated actinomycin D concentration 0.409 ng/mL corresponding to 4.09 ng/g due to tissue homogenization at 0.1 g/mL). The intensity of the blanks was normalized to the value of the analyte peak in the LLOQ chromatogram while the intensity in the remaining chromatograms was normalized to the analyte peak with the IS and analyte transition processed independently. Full article ">Figure 3
Plasma concentration–time profiles of intravenous actinomycin D (0.5 mg/kg) in three mice. Full article ">Figure 4
(A) Actinomycin D showed nonspecific binding to the microdialysate equipment, which can be reduced by adding bovine serum albumin to the Ringer solution. (B) Actinomycin D concentration detected in in vivo cerebral microdialysis samples of a non-dosed mouse after prior in vitro calibration of the microdialysis probe with actinomycin D. Full article ">Figure 5
Schematic workflow of the methodology for determining the central nervous system (CNS) drug disposition of actinomycin D (Act D). Despite the use of actinomycin C (Act C) for probe calibration and avoiding nonspecific binding of actinomycin D to the microdialysis equipment, only whole brain tissue measurements were suitable for measurement of the drug´s brain disposition. Created with BioRender (Biorender AG, Münchwilen, Switzerland). Full article ">Figure 6
(A) Concentration-dependent effect of actinomycin D on the proliferation of the P-gp over-expressing cell line L-MDR1 and the corresponding parental cell line LLC-PK1. Each curve depicts the results of four experiments with each concentration tested in octuplet. Data are expressed as the mean ± SD for n = 32 wells. The IC50 in L-MDR1 cells was 2.4 ± 0.6 µM and in LLC-PK1 cells 0.021 ± 0.006 µM (>100-fold difference). (B) Time-dependent uptake of 0.01 µM actinomycin D in the P-gp over-expressing cell line L-MDR1 in comparison to the parental cell line LLC-PK1. Each data point depicts the mean of a duplicate determination. Full article ">

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