Pharmaceutics

21 pages, 3719 KiB

Open AccessArticle

Cyclin-Dependent Kinase 8 Represents a Positive Regulator of Cytomegalovirus Replication and a Novel Host Target for Antiviral Strategies

by Debora Obergfäll, Markus Wild, Mona Sommerer, Malena Barillas Dahm, Jintawee Kicuntod, Julia Tillmanns, Melanie Kögler, Josephine Lösing, Kishore Dhotre, Regina Müller, Christina Wangen, Sabrina Wagner, Quang V. Phan, Lüder Wiebusch, Katarína Briestenská, Jela Mistríková, Lauren Kerr-Jones, Richard J. Stanton, Sebastian Voigt, Friedrich Hahn and Manfred Marschall Show full author list Hide full author list

Pharmaceutics 2024, 16(9), 1238; https://doi.org/10.3390/pharmaceutics16091238 - 23 Sep 2024

Viewed by 1248

Abstract

Background. Cyclin-dependent kinase 8 (CDK8) is a multifaceted regulator and represents a catalytic component of the transcriptional Mediator complex. CDK8 activity, on the one hand, increases transcriptional elongation by the recruitment of Mediator/super elongation complexes, but, on the other hand, negatively regulates [...] Read more.

Background. Cyclin-dependent kinase 8 (CDK8) is a multifaceted regulator and represents a catalytic component of the transcriptional Mediator complex. CDK8 activity, on the one hand, increases transcriptional elongation by the recruitment of Mediator/super elongation complexes, but, on the other hand, negatively regulates CDK7-controlled transcriptional initiation through inactivating cyclin H phosphorylation. Recently, these combined properties of CDK8 have also suggested its rate-limiting importance for herpesviral replication. Objectives. In this paper, we focused on human cytomegalovirus (HCMV) and addressed the question of whether the pharmacological inhibition or knock-down of CDK8 may affect viral replication efficiency in cell culture models. Methods. A number of human and animal herpesviruses, as well as non-herpesviruses, were used to analyze the importance of CDK8 for viral replication in cell culture models, and to assess the antiviral efficacy of CDK8 inhibitors. Results. Using clinically relevant CDK8 inhibitors (CCT-251921, MSC-2530818, and BI-1347), HCMV replication was found strongly reduced even at nanomolar drug concentrations. The EC₅₀ values were consistent for three different HCMV strains (i.e., AD169, TB40, and Merlin) analyzed in two human cell types (i.e., primary fibroblasts and astrocytoma cells), and the drugs comprised a low level of cytotoxicity. The findings highlighted the following: (i) the pronounced in vitro SI values of anti-HCMV activity obtained with CDK8 inhibitors; (ii) a confirmation of the anti-HCMV efficacy by CDK8–siRNA knock-down; (iii) a CDK8-dependent reduction in viral immediate early, early, and late protein levels; (iv) a main importance of CDK8 for viral late-stage replication; (v) several mechanistic aspects, which point to a strong impact on viral progeny production and release, but a lack of CDK8 relevance for viral entry or nuclear egress; (vi) a significant anti-HCMV drug synergy for combinations of inhibitors against host CDK8 and the viral kinase vCDK/pUL97 (maribavir); (vii) finally, a broad-spectrum antiviral activity, as seen for the comparison of selected α-, β-, γ-, and non-herpesviruses. Conclusions. In summary, these novel data provide evidence for the importance of CDK8 as a positive regulator of herpesviral replication efficiency, and moreover, suggest its exploitability as an antiviral target for novel strategies of host-directed drug development. Full article

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Anti-HCMV activity was demonstrated for three CDK8-inhibitory developmental small molecules: (A) CCT-251921, (B) MSC-2530818, and (C) BI-1347. The assessment utilized a GFP replication assay employing HCMV AD169-GFP for infecting HFFs. The compounds were administered immediately p.i., starting at a concentration of 50 nM, followed by eight consecutive five-fold dilution steps. Cells were fixed at 7 d p.i. for quantitative GFP fluorometry. Additionally, cell viability in uninfected cells was determined through NRA (curves in orange). The presented values represent the mean ± SD of triplicate (NRA) or quadruplicate (GFP), with the data representing one experiment out of at least three independent replicates. Full article ">Figure 2
Anti-HCMV activity was demonstrated for three CDK8-inhibitory developmental small molecules: For Merlin-GFP in HFF cells (A) CCT-251921, (B) MSC-2530818, and (C) BI-1347, and AD169-GFP in U373 cells (D) CCT-251921, (E) MSC-2530818, and (F) BI-1347. The experiment utilizes a GFP replication assay with HCMV AD169-GFP or Merlin-GFP to infect HFF or U373 cells. The compounds were administered immediately post-infection (p.i.), beginning at a concentration of 50 nM, followed by eight five-fold dilution steps. Cells were fixed at 7 days post-infection (d p.i.) for quantitative GFP fluorometry. Additionally, cell viability in uninfected cells was assessed using the NRA (curves in orange). The values presented are the mean ± SD of triplicate (NRA) or quadruplicate (GFP) measurements, with data representing one experiment out of at least three independent replicates. Full article ">Figure 3
Analysis of the antiviral effect of the CCT CDK8 inhibitor. HFFs were cultivated in 6-well plates and infected with HCMV AD169 at MOI of 0.1. Infected cells were treated with 30 nM, 3 nM or 0.3 nM CCT. Mock-infected cells and DMSO-treated infected cells were used as control samples. Cells were harvested at 3 d (A), 5 d (B), and 7 d p.i. (C) for the preparation of total lysates, to be analyzed by SDS-PAGE/Wb procedures. Wb antibody staining was performed for viral IE1p72, pUL44, and pp28, thus representing viral immediate early (IE), early (E), and late (L) marker proteins, respectively. In addition, cellular CDK8 (drug target) as well as β-actin (house-keeping protein and loading control) were stained in parallel. Full article ">Figure 4
Analysis of transfection-mediated intracellular CDK8-specific siRNA knock-down (KD) in primary fibroblasts. (A) The CDK8-specific siRNA KD was specifically reduced, under slightly optimized conditions (Lipofectamine™ RNAiMAX), using the siRNAs CDK8 100 at a final concentration of 10 pmol/well. HFFs were cultivated in 6-well plates and infected with HCMV AD169 at MOI of 0.1 at two d post-siRNA transfection (see KD effect highlighted by dashed box). As control samples, mock-infected cells, mock-transfected (no siRNA) cells, a non-specific siRNA (scrambled), and a GAPDH-specific siRNA were used. Cells were harvested at 7 d p.i. for the preparation of total lysates, to be analyzed by SDS-PAGE/Wb procedures. (B) Densitometric analysis was performed in quadruplicate measurements (SDS-PAGE/Wbs in duplicate, densitometry in duplicate) using AIDA image analyzer, and statistical evaluation was conducted using ANOVA followed by Bonferroni’s correction method for multiple comparison (** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001). Full article ">Figure 5
Mechanistic characteristics of the antiviral activity of CDK8 inhibitors. (A) An analysis of virus entry in the presence of CDK8 inhibitors was performed by comparing pre-post-incubation and post-incubation conditions of drug treatment. HFFs were infected with recombinant HCMV AD169 expressing an HA-tagged pUL50 (AD169-UL50-HA) at MOI of 0.01. For vCDK and CDK8 inhibitor treatments, two different setups were used. One set of cells was treated with MBV (0.5 µM and 1.5 µM) or CCT (5 nM and 15 nM) before, during, and after infection (pre-post). The other set was exclusively treated 90 min after virus absorption (post). Western blot staining was performed for IE1p72, pUL50-HA, pUL53, MCP, and CDK8. (B) The typical nuclear rim localization of viral NEC proteins was analyzed under CDK8 inhibitor treatment. HFFs were cultivated in 6-well plates on cover slips, and used for infection with HCMV AD169-UL50-HA at MOI of 0.05. After 90 min of virus absorption, cells were treated with 0.5 µM MBV, 5 nM CCT, 2 nM MSC, or 0.7 nM BI. Infected cells were fixed at 7 d p.i., and IF staining was performed for viral pUL50-HA and pUL53 to be analyzed by confocal imaging. Counterstaining of the nuclei (DAPI) is indicated, and a merge of pUL50-HA and pUL53 signals is provided on the right. (C) The reduction in virus progeny production and release was quantitated under CDK8 inhibitor treatment. For this purpose, HFFs were infected with AD169-UL50-HA at MOI of 0.01 and treated with 5 nM CCT, 2 nM MSC, 0.7 nM BI, or DMSO in the control. Supernatant samples were taken at 2, 4, 7, 9, 11, and 14 d p.i., to perform virus-specific qPCR analysis. Statistical evaluation was conducted using an ANOVA followed by Bonferroni’s correction method for multiple comparison (* p ≤ 0.05, ** p ≤ 0.01, **** p ≤ 0.0001). Full article ">Figure 6
Antiviral drug combination treatment using Loewe additivity fixed-dose assay. The analysis included the (A) combination of MBV + CCT-251921, (B) combination of MBV + MSC-2530818, and (C) combination of MBV + BI-1347. A modified protocol of the HCMV-GFP replication system was used, and cells were treated with either single compounds, compound combinations (MBV + CDK8 inhibitor CCT, MSC, or BI, at the concentration ratio of 1:1000), or a solvent control. Antiviral efficacy (mean of quadruplicate measurements of biological duplicates) was expressed as a percentage of the solvent control and analyzed using the CompuSyn software. Only experiments with an r value > 0.90 and EC50 values near previously determined concentrations were accepted. Full article ">

28 pages, 1603 KiB

Open AccessReview

Alternative Cancer Therapeutics: Unpatentable Compounds and Their Potential in Oncology

by Dmitriy Ovcharenko, Dmitry Mukhin and Galina Ovcharenko

Pharmaceutics 2024, 16(9), 1237; https://doi.org/10.3390/pharmaceutics16091237 - 23 Sep 2024

Viewed by 1808

Abstract

Cancer remains a leading cause of death globally. Cancer patients often seek alternative therapies in addition to, or instead of, conventional treatments like chemotherapy, radiation, and surgery. The progress in medical advancements and early detection provides more treatment options; however, the development of [...] Read more.

Cancer remains a leading cause of death globally. Cancer patients often seek alternative therapies in addition to, or instead of, conventional treatments like chemotherapy, radiation, and surgery. The progress in medical advancements and early detection provides more treatment options; however, the development of cancer drugs requires a significant amount of time, demands substantial investments, and results in an overall low percent of regulatory approval. The complex relationship between patent protection and pharmaceutical innovation complicates cancer drug development and contributes to high mortality rates. Adjusting patent criteria for alternative cancer therapeutics could stimulate innovation, enhance treatment options, and ultimately improve outcomes for cancer patients. This article explores the potential of alternative cancer therapeutics, chemopreventive agents, natural products, off-patent drugs, generic unpatentable chemicals, and repurposed drugs in cancer treatment, emphasizing the mechanisms and therapeutic potential of these unconventional compounds as combinatorial cancer therapies. The biological pathways, therapeutic effects, and potential to enhance existing therapies are reviewed, demonstrating their cost-effective and accessible options as adjuvant cancer therapies. Full article

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19 pages, 4062 KiB

Open AccessArticle

Molded Round Window Niche Implant as a Dexamethasone Delivery System in a Cochlear Implant-Trauma Animal Model

by Chunjiang Wei, Ziwen Gao, Robert Mau, Thomas Eickner, Gabor Jüttner, Nicklas Fiedler, Hermann Seitz, Thomas Lenarz and Verena Scheper

Pharmaceutics 2024, 16(9), 1236; https://doi.org/10.3390/pharmaceutics16091236 - 23 Sep 2024

Viewed by 3622

Abstract

Background: Preserving residual hearing after cochlear implant (CI) surgery remains a crucial challenge. The application of dexamethasone (DEX) has been proven to positively affect residual hearing. To deliver DEX in a localized and controlled way, a round window niche implant (RNI), allowing drug [...] Read more.

Background: Preserving residual hearing after cochlear implant (CI) surgery remains a crucial challenge. The application of dexamethasone (DEX) has been proven to positively affect residual hearing. To deliver DEX in a localized and controlled way, a round window niche implant (RNI), allowing drug diffusion via the round window membrane into the cochlea, may be used. To prove this concept, an RNI for guinea pigs as a CI-trauma model was manufactured by molding and tested for its drug release in vitro and biological effects in vivo. Methods: The RNIs were molded using silicone containing 10% DEX. Release was analyzed over time using high-performance liquid chromatography (HPLC). Fourteen adult guinea pigs were randomly assigned to two groups (CI or CI + RNI group). All animals received a unilateral CI electrode insertion trauma followed by CI insertion. The CI + RNI group was additionally implanted with an RNI containing 10% DEX. Animals were followed up for 4 weeks. Acoustically evoked auditory brainstem response and impedance measurement, micro-computed tomography (µCT) imaging, and histology were performed for evaluation. Results: DEX was released for more than 250 days in vitro, with an initial burst followed by a slower release over time. Comparing the hearing threshold shift (from day 0 to day 28) of the CI and CI + RNI groups, significant differences were observed at 32 and 40 kHz. The impedance shift at basal contacts was lower in the CI + RNI group than in the CI group. Moreover, the fibrosis in the lower basal turn was reduced in the CI + RNI group in contrast to the CI group. Conclusions: The RNI containing 10% DEX has anti-inflammatory potential concerning fibrosis inhibition and has beneficial effects on hearing preservation at high frequencies. Full article

(This article belongs to the Special Issue Site-Specific Drug Delivery: Formulation Strategies and Regulatory Challenges)

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12 pages, 1289 KiB

Open AccessArticle

Analysis of Lipophilicity and Pharmacokinetic Parameters of Dipyridothiazine Dimers with Anticancer Potency

by Emilia Martula, Beata Morak-Młodawska, Małgorzata Jeleń and Patrick Nwabueze Okechukwu

Pharmaceutics 2024, 16(9), 1235; https://doi.org/10.3390/pharmaceutics16091235 - 23 Sep 2024

Viewed by 656

Abstract

Lipophilicity is an essential parameter of a compound that determines the solubility and pharmacokinetic properties that determine the transport of the drug to the molecular target. Dimers of dipyridothiazines are diazaphenothiazine derivatives exhibiting diverse anticancer potential in vitro, which is related to their [...] Read more.

Lipophilicity is an essential parameter of a compound that determines the solubility and pharmacokinetic properties that determine the transport of the drug to the molecular target. Dimers of dipyridothiazines are diazaphenothiazine derivatives exhibiting diverse anticancer potential in vitro, which is related to their affinity for histone deacetylase. In this study, the lipophilicity of 16 isomeric dipyridothiazine dimers was investigated theoretically and experimentally by reversed-phase thin-layer chromatography (RP-TLC) in an acetone–TRIS buffer (pH = 7.4). The relative lipophilicity parameter R_M0 and specific hydrophobic surface area b were significantly intercorrelated, showing congeneric classes of dimers. The parameter R_M0 was transformed into parameter logP_TLC by use of the calibration curve. Molecular descriptors, ADMET parameters and probable molecular targets were determined in silico for analysis of the pharmacokinetic profile of the tested compounds showing anticancer activity. The analyzed compounds were tested in the context of Lipinski’s rule of five, Ghose’s rule and Veber’s rule, confirming their bioavailability. Full article

(This article belongs to the Special Issue Role of Pharmacokinetics in Drug Development and Evaluation)

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14 pages, 2300 KiB

Open AccessArticle

Glioblastoma Multiforme: Sensitivity to Antimicrobial Peptides LL-37 and PG-1, and Their Combination with Chemotherapy for Predicting the Overall Survival of Patients

by Alexander N. Chernov, Sofia S. Skliar, Alexander V. Kim, Anna Tsapieva, Sarng S. Pyurveev, Tatiana A. Filatenkova, Marina V. Matsko, Sergey D. Ivanov, Olga V. Shamova and Alexander N. Suvorov

Pharmaceutics 2024, 16(9), 1234; https://doi.org/10.3390/pharmaceutics16091234 - 22 Sep 2024

Viewed by 1292

Abstract

Background/Objectives: Glioblastomas (GBMs) are the most malignant and intractable of all cancers, with an unfavorable clinical prognosis for affected patients. The objective was to analyze the sensitivity of GBM cells to the antimicrobial peptides (AMPs) cathelicidin (LL-37) and protegrin-1 (PG-1), both alone and [...] Read more.

Background/Objectives: Glioblastomas (GBMs) are the most malignant and intractable of all cancers, with an unfavorable clinical prognosis for affected patients. The objective was to analyze the sensitivity of GBM cells to the antimicrobial peptides (AMPs) cathelicidin (LL-37) and protegrin-1 (PG-1), both alone and in combination with chemotherapy, to predict overall survival (OS) in the patients. Methods: The study was conducted on 27 GBM patients treated in the neurosurgical department of the Almazov Medical Research Centre (Saint Petersburg, Russia) from 2021 to 2024. The cytotoxic effects of chemotherapy, AMPs, and their combinations on brain tumor cells were assessed by an MTT assay using a 50% inhibitory concentration (IC₅₀). Results: In GBM cells from the patients, LL-37 and PG-1 exhibited strong anticancer effects, surpassing those of chemotherapy drugs. These LL-37 and PG-1 anticancer effects were associated with a statistically significant increase in life expectancy and OS in GBM patients. These findings were confirmed by experiments on rats with C6 glioma, where the intranasal administration of LL-37 (300 μM) and PG-1 (600 μM) increased the life expectancy of the animals to 69 and 55 days, respectively, compared to 24 days in the control group (HR = 4.139, p = 0.0005; HR = 2.542, p = 0.0759). Conclusions: Additionally, the combination of LL-37 and PG-1 with chemotherapy drugs showed that a high IC₅₀ of LL-37 with cisplatin (cutoff > 800 μM) in GBM cells was associated with increased life expectancy (19 vs. 5 months, HR = 4.708, p = 0.0101) and OS in GBM patients. These combinations could be used in future GBM treatments. Full article

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Overall survival of GBM patients, based on high and low IC50 levels of chemotherapy drugs. The OS of GBM patients was assessed according to their IC50 levels for various chemotherapy drugs: (A) carboplatin, (B) cisplatin, (C) doxorubicin, (D) etoposide, (E) temozolomide. The analysis was conducted using the Mantel–Cox test (χ2), with the following abbreviations: CARB (carboplatin), CIS (cisplatin), DOX (doxorubicin), ETO (etoposide), and TMZ (temozolomide). * Statistically significant (p < 0.05) differences high and low levels IC50 between groups. Full article ">Figure 2
Overall survival of GBM patients, based on high and low IC50 levels of (A) LL-37 and its combinations with chemotherapy drugs: (B) doxorubicin, (C) carboplatin, (D) cisplatin, (E) etoposide, (F) temozolomide. Mantel–Cox test, χ2. * Statistically significant (p < 0.05) differences high and low levels IC50 between groups. Full article ">Figure 3
Overall survival of GBM patients based on high and low IC50 levels of (A) PG-1 and its combinations with chemotherapy drugs: (B) doxorubicin, (C) carboplatin, (D) cisplatin, (E) etoposide, (F) temozolomide. Mantel–Cox test, χ2. * Statistically significant (p < 0.05) differences high and low levels IC50 between groups. Full article ">Figure 4
Overall survival of Wistar rats with C6 glioma following the administration of (A) LL-37 and (B) PG-1. Tumor volume following the administration of (C) LL-37 and (D) PG-1. The survival analysis was performed using the Mantel–Cox test with the χ2 values indicated. ** Statistically significant (p < 0.01) and **** (p < 0.0001) differences between tumor volume and overall survival rats under LL-37 administration from control group. Full article ">

23 pages, 4533 KiB

Open AccessArticle

Exploring Cationic Guar Gum: Innovative Hydrogels and Films for Enhanced Wound Healing

by Kamila Gabrieli Dallabrida, Willer Cezar Braz, Crisleine Marchiori, Thainá Mayer Alves, Luiza Stolz Cruz, Giovanna Araujo de Morais Trindade, Patrícia Machado, Lucas Saldanha da Rosa, Najeh Maissar Khalil, Fabiane Gomes de Moraes Rego, André Ricardo Fajardo, Luana Mota Ferreira, Marcel Henrique Marcondes Sari and Jéssica Brandão Reolon

Pharmaceutics 2024, 16(9), 1233; https://doi.org/10.3390/pharmaceutics16091233 - 22 Sep 2024

Cited by 1 | Viewed by 1177

Abstract

Background/Objectives: This study developed and characterized hydrogels (HG-CGG) and films (F-CGG) based on cationic guar gum (CGG) for application in wound healing. Methods: HG-CGG (2% w/v) was prepared by gum thickening and evaluated for pH, stability, spreadability, and viscosity. F-CGG [...] Read more.

Background/Objectives: This study developed and characterized hydrogels (HG-CGG) and films (F-CGG) based on cationic guar gum (CGG) for application in wound healing. Methods: HG-CGG (2% w/v) was prepared by gum thickening and evaluated for pH, stability, spreadability, and viscosity. F-CGG was obtained using an aqueous dispersion of CGG (6% w/v) and the solvent casting method. F-CGG was characterized for thickness, weight uniformity, morphology, mechanical properties, hydrophilicity, and swelling potential. Both formulations were evaluated for bioadhesive potential on intact and injured porcine skin, as well as antioxidant activity. F-CGG was further studied for biocompatibility using hemolysis and cell viability assays (L929 fibroblasts), and its wound-healing potential by the scratch assay. Results: HG-CGG showed adequate viscosity and spreadability profiles for wound coverage, but its bioadhesive strength was reduced on injured skin. In contrast, F-CGG maintained consistent bioadhesive strength regardless of skin condition (6554.14 ± 540.57 dyne/cm² on injured skin), presenting appropriate mechanical properties (flexible, transparent, thin, and resistant) and a high swelling capacity (2032 ± 211% after 6 h). F-CGG demonstrated superior antioxidant potential compared to HG-CGG (20.50 mg/mL ABTS+ radical scavenging activity), in addition to exhibiting low hemolytic potential and no cytotoxicity to fibroblasts. F-CGG promoted the proliferation of L929 cells in vitro, supporting wound healing. Conclusions: Therefore, CGG proved to be a promising material for developing formulations with properties suitable for cutaneous use. F-CGG combines bioadhesion, antioxidant activity, biocompatibility, cell proliferation, and potential wound healing, making it promising for advanced wound treatment. Full article

(This article belongs to the Special Issue Research on the Development of Nano-Based Polymeric Films for Drugs and Their Derivatives)

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49 pages, 2873 KiB

Open AccessReview

Unlocking the Potential of Silver Nanoparticles: From Synthesis to Versatile Bio-Applications

by Ahmad Almatroudi

Pharmaceutics 2024, 16(9), 1232; https://doi.org/10.3390/pharmaceutics16091232 - 21 Sep 2024

Cited by 2 | Viewed by 1508

Abstract

Silver nanoparticles (AgNPs) are leading the way in nanotechnological innovation, combining the captivating properties of silver with the accuracy of nanoscale engineering, thus revolutionizing material science. Three main techniques arise within the alchemical domains of AgNP genesis: chemical, physical, and biological synthesis. Each [...] Read more.

Silver nanoparticles (AgNPs) are leading the way in nanotechnological innovation, combining the captivating properties of silver with the accuracy of nanoscale engineering, thus revolutionizing material science. Three main techniques arise within the alchemical domains of AgNP genesis: chemical, physical, and biological synthesis. Each possesses its distinct form of magic for controlling size, shape, and scalability—key factors necessary for achieving expertise in the practical application of nanoparticles. The story unravels, describing the careful coordination of chemical reduction, the environmentally sensitive charm of green synthesis utilizing plant extracts, and the precise accuracy of physical techniques. AgNPs are highly praised in the field of healthcare for their powerful antibacterial characteristics. These little warriors display a wide-ranging attack against bacteria, fungi, parasites, and viruses. Their critical significance in combating hospital-acquired and surgical site infections is highly praised, serving as a beacon of hope in the fight against the challenging problem of antibiotic resistance. In addition to their ability to kill bacteria, AgNPs are also known to promote tissue regeneration and facilitate wound healing. The field of cancer has also observed the adaptability of AgNPs. The review documents their role as innovative carriers of drugs, specifically designed to target cancer cells with accuracy, minimizing harm to healthy tissues. Additionally, it explores their potential as cancer therapy or anticancer agents capable of disrupting the growth of tumors. In the food business, AgNPs are utilized to enhance the durability of packing materials and coatings by infusing them with their bactericidal properties. This results in improved food safety measures and a significant increase in the duration that products can be stored, thereby tackling the crucial issue of food preservation. This academic analysis recognizes the many difficulties that come with the creation and incorporation of AgNPs. This statement pertains to the evaluation of environmental factors and the effort to enhance synthetic processes. The review predicts future academic pursuits, envisioning progress that will enhance the usefulness of AgNPs and increase their importance from being new to becoming essential within the realms of science and industry. Besides, AgNPs are not only a subject of scholarly interest but also a crucial component in the continuous effort to tackle some of the most urgent health and conservation concerns of contemporary society. This review aims to explore the complex process of AgNP synthesis and highlight their numerous uses, with a special focus on their growing importance in the healthcare and food business sectors. This review invites the scientific community to explore the extensive possibilities of AgNPs in order to fully understand and utilize their potential. Full article

(This article belongs to the Special Issue Novel Micro/Nanomaterials Based Drug Delivery Systems for Theranostic Applications)

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11 pages, 922 KiB

Open AccessArticle

Automated Production of [⁶⁸Ga]Ga-Desferrioxamine B on Two Different Synthesis Platforms

by Martin Kraihammer, Miloš Petřík, Christine Rangger, Michael Gabriel, Hubertus Haas, Bernhard Nilica, Irene Virgolini and Clemens Decristoforo

Pharmaceutics 2024, 16(9), 1231; https://doi.org/10.3390/pharmaceutics16091231 - 21 Sep 2024

Viewed by 751

Abstract

Background/Objectives: PET imaging of bacterial infection could potentially provide added benefits for patient care through non-invasive means. [⁶⁸Ga]Ga-desferrioxamine B—a radiolabelled siderophore—shows specific uptake by human-pathogenic bacteria like Staphylococcus aureus or Pseudomonas aeruginosa and sufficient serum stability for clinical application. In this [...] Read more.

Background/Objectives: PET imaging of bacterial infection could potentially provide added benefits for patient care through non-invasive means. [⁶⁸Ga]Ga-desferrioxamine B—a radiolabelled siderophore—shows specific uptake by human-pathogenic bacteria like Staphylococcus aureus or Pseudomonas aeruginosa and sufficient serum stability for clinical application. In this report, we present data for automated production of [⁶⁸Ga]Ga-desferrioxamine B on two different cassette-based synthesis modules (Modular-Lab PharmTracer and GRP 3V) utilising commercially obtainable cassettes together with a licensed ⁶⁸Ge/⁶⁸Ga radionuclide generator. Methods: Quality control, including the determination of radiochemical purity, as well as a system suitability test, was set up via RP-HPLC on a C18 column. The two described production processes use an acetic acid/acetate buffer system with ascorbic acid as a radical scavenger for radiolabelling, yielding ready-to-use formulations with sufficient activity yield. Results: Batch data analysis demonstrated radiochemical purity of >95% by RP-HPLC combined with ITLC and excellent stability up to 2 h after synthesis. Specifications for routine production were set up and validated with four masterbatches for each synthesis module. Conclusions: Based on this study, an academic clinical trial for imaging of bacterial infection was initiated. Both described synthesis methods enable automated production of [⁶⁸Ga]Ga-desferrioxamine B in-house with high reproducibility for clinical application. Full article

(This article belongs to the Special Issue Advances in Radiopharmaceuticals for Disease Diagnoses and Therapy)

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17 pages, 8494 KiB

Open AccessArticle

Enhancing Radiotherapy Sensitivity in Prostate Cancer with Lentinan-Functionalized Selenium Nanoparticles: Mechanistic Insights and Therapeutic Potential

by Yani Zou, Helin Xu, Xiu Wu, Xuesong Liu and Jianfu Zhao

Pharmaceutics 2024, 16(9), 1230; https://doi.org/10.3390/pharmaceutics16091230 - 21 Sep 2024

Viewed by 1176

Abstract

Radiation therapy is a cornerstone of prostate cancer (PCa) treatment. However, its limited tumor sensitivity and severe side effects restrict its clinical utility. Lentinan-functionalized selenium nanoparticles (LET-SeNPs) have shown promise in enhancing radiotherapy sensitivity and exhibiting antitumor activity. In this study, we investigated [...] Read more.

Radiation therapy is a cornerstone of prostate cancer (PCa) treatment. However, its limited tumor sensitivity and severe side effects restrict its clinical utility. Lentinan-functionalized selenium nanoparticles (LET-SeNPs) have shown promise in enhancing radiotherapy sensitivity and exhibiting antitumor activity. In this study, we investigated the radiotherapy sensitization mechanism of LET-SeNPs in PCa. Our results demonstrate that the combination of LET-SeNPs and X-ray therapy (4 Gy) significantly inhibited the growth and colony formation of PCa cells by inducing apoptosis, surpassing the effects of individual treatments. This combined approach modulated DNA damage through the p53, MAPK (mitogen-activated protein kinase), and AKT pathways. Furthermore, LET-SeNPs increased PC3 cell sensitivity to X-ray-induced apoptosis by downregulating TrxR (Thioredoxin reductase) expression and inducing reactive oxygen species (ROS) overproduction, thereby activating mitochondria-mediated apoptosis signaling pathways. Additionally, LET-SeNPs regulated PARP (poly (ADP-ribose) polymerase) to prevent DNA damage repair. In vivo studies confirmed that the combination treatment inhibited PCa growth by synergistically activating the p53 pathway to induce cell apoptosis. These findings highlight LET-SeNPs’ potential as a radiotherapy sensitizer and suggest that combining LET-SeNPs with X-ray therapy could be a promising strategy for clinical application, leveraging selenium-modified nanoparticles’ antitumor effects. Full article

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The survival rate of PC3 cells was studied by examining the effects of various levels of LET-SeNPs and doses of X-ray. (A) Process flow diagram of LET-SeNP preparation. (B,C) TEM image of LET-SeNPs. (D) Stability of LET-SeNPs in PBS, DMEM, and FBS. (E) Zeta potential of LET-SeNPs. (F) Cytotoxicity of LET-SeNPs (16, 32, and 64 μM) with X-ray (0, 2, and 4 Gy) on PC3 cells. (G) PC3 cells were exposed to X-ray (0, 2, and 4 Gy) irradiation followed by incubation for 72 h. (H) LET-SeNPs combined with X-ray (2 and 4 Gy) enhances the anticancer efficacy. An isobologram was used to analyze the combined effect of X-ray (2 and 4 Gy) radiation and LET-SeNPs (16.6 μM, 5.6 μM) on inhibiting the growth of PC3 cells. (I) The viability of PC3 cells was assessed after exposure to X-ray (0, 2, 4,8, 16, and 32 Gy). Each value represents means ± SD (n = 3). Full article ">Figure 2
The combination of LET-SeNPs and X-ray (4 Gy) suppressed the clonogenic ability of PC3 cells. (A) The combination of various levels of LET-SeNPs (1, 2, and 4 μM) and X-ray (4 Gy) affected the ability of PC3 cells to form colonies and survive. (B) Colony assay of PC3 cells was assessed when treated with varying doses of LET-SeNPs (1, 2, and 4 μM) in combination with X-ray (4 Gy) radiation. Each value represents mean ± SD (n = 3). ** p < 0.01, *** p < 0.001. Full article ">Figure 3
Cell cycle arrest and apoptosis analysis after treatment with LET-SeNPs combined with X-ray (4 Gy) radiotherapy. (A) Flow cytometric analysis of PC3 cells under treatment with or without different concentrations of LET-SeNPs (1, 2, and 4 μM) and X-ray (4 Gy). (C) Flow cytometry apoptosis of PC3 cells treatment with or without different concentrations of LET-SeNPs (1, 2, and 4 μM) and X-ray (4 Gy). (B,D) Quantitative analysis of PC3 cell cycle arrest and apoptosis. Full article ">Figure 4
After 2 h of treatment with LET-SeNPs combined with X-ray radiotherapy (4 Gy), the mitochondrial membrane potential and ROS levels in PC3 cells were measured. (A) Flow cytometric analysis of the mitochondrial membrane potential of PC3 cells after treatment with various concentrations of LET-SeNPs (1, 2, and 4 μM) and X-ray (4 Gy). (B) JC-1 fluorescence detected by fluorescent microscope after treatment with varying concentrations of LET-SeNPs (4 μM) and X-ray (4 Gy). (C) ROS levels of PC3 cells treated by LET-SeNPs (1, 2, and 4 μM) and X-ray (4 Gy). (D) Fluorescence imaging in PC3 cells marked with DCF. Full article ">Figure 5
Activation of apoptotic signaling pathways by LET-SeNPs and X-ray (4 Gy). (A) LET-SeNPs combined with X-ray (4 Gy) enhanced p53 signal pathway. (B) LET-SeNPs combined with X-ray (4 Gy) effects the expression of MAPKs and AKT pathways in PC3 cells, and the expression levels of TrxR and PARP. (C) Activation of caspase-3 pathway in different groups. *** p < 0.001, **** p < 0.0001. (D) LET-SeNPs combined with X-ray (4 Gy) proposed signal pathways of apoptosis. Full article ">Figure 6
In vivo anticancer effects of LET-SeNPs and X-ray tested in nude mice. (A,B) The growth curves and body weight of tumors on mice treated with LET-SeNPs and X-ray. (C,D) The images of representative mice and tumors in each group. (E) Microscopic pictures of tumor sections stained with H&E and immunohistochemical p-p53 from various groups. Full article ">Figure 7
Major organs and blood biochemical indexes were evaluated. (A) Heart, liver, spleen, lung, and kidney in 4 groups were assessed for potential toxicity by H&E staining. (B,C) AST and UA were analyzed to evaluate systemic toxicity. Full article ">Figure 8
Schematic diagram of bilateral tumor-bearing mouse model. (A,B) After 21 days of treatment with LET-SeNPs and X-ray (4 Gy), images of mice and tumors were taken. The left sides of the mice were treated with LET-SeNPs (2 mg/kg), while the right sides were treated with LET-SeNPs (2 mg/kg) in combination with X-ray (4 Gy). (C) Microscope images of H&E and p-p53 immunohistochemical staining of tumors from nude mice with tumors on both sides. Full article ">Figure 9
The proposed approach includes the utilization of functionalized LET-SeNPs to increase the efficacy of radiotherapy on prostate cancer PC3 cells. The joint therapy efficiently induces excessive ROS production within cells to control the p53-related DNA damage apoptosis signaling pathway and phosphorylation of MAPK and AKT. (By Figdraw, <a href="http://www.figdraw.com" target="_blank">www.figdraw.com</a>). Full article ">

15 pages, 2077 KiB

Open AccessArticle

Oral Gels as an Alternative to Liquid Pediatric Suspensions Compounded from Commercial Tablets

by Monika Trofimiuk, Małgorzata Sznitowska and Katarzyna Winnicka

Pharmaceutics 2024, 16(9), 1229; https://doi.org/10.3390/pharmaceutics16091229 - 20 Sep 2024

Viewed by 876

Abstract

The aim of the study was to propose pharmacy-compounded oral gels as a new and alternative dosage form that is attractive to children as having a better masking taste than syrups and reducing the risk of spilling. The application and physical properties of [...] Read more.

The aim of the study was to propose pharmacy-compounded oral gels as a new and alternative dosage form that is attractive to children as having a better masking taste than syrups and reducing the risk of spilling. The application and physical properties of the gels prepared with cellulose derivatives (hydroxyethylcellulose and carmellose sodium) or carbomers were evaluated. The results of the study showed the most suitable consistency, viscosity, and organoleptic properties for gels prepared with carbomer and cellulose derivatives at concentrations of 0.75% and 2.0%, respectively. The microbial stability of the gels was guaranteed by the use of methylparaben and potassium sorbate. VAL (valsartan) and CC (candesartan cilexetil) tablets, often used off-label in children, were pulverized and suspended in the hydrogel bases, resulting in final drug concentrations of 4 mg/g and 1 mg/g, respectively. There was no significant change in viscosity and consistency parameters when the pulverized tablets were added, and only small changes in viscosity and consistency were observed during 35 days of storage, especially in the gels with sodium carmellose and candesartan. On the basis of the drug assay, an expiry date of 25 °C was recommended: 35 days for valsartan and 14 days for candesartan preparations. Full article

(This article belongs to the Special Issue Extemporaneous Formulations: Filling the Gap in the Pharmaceutical Industry with Personalized Medicines)

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The curve and mechanical parameters of hydrogels were tested in a texture analyzer with a downstream and upstream movement of the probe. Full article ">Figure 2
Hydrogels are prepared from valsartan (VAL) and candesartan cilexetil (CC) tablets using hydrogel carriers with hydroxyethylcellulose (HEC), carmellose sodium (CMC), and carbomer (CAR). Full article ">Figure 3
The mechanical properties of hydrogels prepared with VAL and CC tablets using hydrogel media (HEC, CMC, and CAR) and stored at 25 °C for 35 days. The mechanical parameters at t0 for the placebo hydrogels are also presented. * Statistical significance was demonstrated after the addition of the tablet mass (p < 0.05). ^ Statistical significance was demonstrated in relation to time t0, (p < 0.05). Full article ">Figure 4
The representative hysteresis loops for HEC, CMC, and CAR gels were recorded for placebo gels at t0 (A) and gels with candesartan cilexetil (B) and valsartan (C) tablets after preparation (t0) and 35 days of storage at 25 °C. Full article ">Figure 5
Chromatograms of VAL (a) and CC (b) in CMC gels: after preparation (t0) and after 14 and 35 days of storage at 25 °C. Full article ">Figure 6
Stability (% of the initial content) of VAL and CC in oral hydrogels prepared with HEC, CMC, and CAR, stored at 25 °C and 4 °C for 35 days. Full article ">

23 pages, 9925 KiB

Open AccessReview

Nanotechnology in Advancing Chimeric Antigen Receptor T Cell Therapy for Cancer Treatment

by Xuejia Kang, Nur Mita, Lang Zhou, Siqi Wu, Zongliang Yue, R. Jayachandra Babu and Pengyu Chen

Pharmaceutics 2024, 16(9), 1228; https://doi.org/10.3390/pharmaceutics16091228 - 20 Sep 2024

Viewed by 1530

Abstract

Chimeric antigen receptor (CAR) T cell therapy has emerged as a groundbreaking treatment for hematological cancers, yet it faces significant hurdles, particularly regarding its efficacy in solid tumors and concerning associated adverse effects. This review provides a comprehensive analysis of the advancements and [...] Read more.

Chimeric antigen receptor (CAR) T cell therapy has emerged as a groundbreaking treatment for hematological cancers, yet it faces significant hurdles, particularly regarding its efficacy in solid tumors and concerning associated adverse effects. This review provides a comprehensive analysis of the advancements and ongoing challenges in CAR-T therapy. We highlight the transformative potential of nanotechnology in enhancing CAR-T therapy by improving targeting precision, modulating the immune-suppressive tumor microenvironment, and overcoming physical barriers. Nanotechnology facilitates efficient CAR gene delivery into T cells, boosting transfection efficiency and potentially reducing therapy costs. Moreover, nanotechnology offers innovative solutions to mitigate cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS). Cutting-edge nanotechnology platforms for real-time monitoring of CAR-T cell activity and cytokine release are also discussed. By integrating these advancements, we aim to provide valuable insights and pave the way for the next generation of CAR-T cell therapies to overcome current limitations and enhance therapeutic outcomes. Full article

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Nanoparticles enhancing CAR-T cell therapy. Nanoparticles (NPs) improve CAR-T cell therapy by facilitating efficient construction, transfection, expansion, and monitoring. NPs aid in gene delivery during transfection, enhance CAR-T cell expansion, and provide real-time monitoring capabilities, addressing challenges in treating cancer. Created with <a href="http://BioRender.com" target="_blank">BioRender.com</a>, accessed on 10 July 2024. Full article ">Figure 2
Illustration of the comparative benefits and challenges of using nanoparticles versus viral vectors in CAR-based therapies, highlighting the potential of nanotechnology to overcome current limitations in safety, efficacy, and cost. Created with <a href="http://BioRender.com" target="_blank">BioRender.com</a>, accessed on 10 July 2024. Full article ">Figure 3
Nanotechnology serves as a versatile tool to enhance the precision and efficacy of CAR-T cell therapies. Specifically, nanotechnology can achieve controlled release of CAR-T cells, improve CAR-T cell penetration, destroy stromal cells, and reduce tumor burden. Additionally, nanotechnology can be used as a carrier to load immunomodulatory factors, cytokines, or associated inhibitors to modulate the tumor microenvironment (TME). Created with <a href="http://BioRender.com" target="_blank">BioRender.com</a>, accessed on 11 July 2024. Full article ">Figure 4
Nanoparticles (NPs) offer promising solutions to alleviate cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS) and mitigate off-target effects and infections in CAR-T cell therapy. Created with <a href="http://BioRender.com" target="_blank">BioRender.com</a>, access 28 July 2024. Full article ">Figure 5
A schematic illustration depicting how cytokine nanosponges (LMNP) suppress overactive macrophages and mitigate systemic cytokine storms, providing treatment for hemophagocytic lymphohistiocytosis (HLH), reprinted with permission [<a href="#B166-pharmaceutics-16-01228" class="html-bibr">166</a>]. Full article ">Figure 6
(A) A machine-learning-assisted microfluidic nanoplasmonic digital immunoassay has been developed for monitoring cytokine storms. © 2021 American Chemical Society. The immunoassay comprises three primary components: (a) the microfluidic immunoassay platform, (b) the nanoplasmonic digital imaging technology, and (c) the machine-learning-based image processing method [<a href="#B180-pharmaceutics-16-01228" class="html-bibr">180</a>]. (B) The detection of cytokines induced by CAR-T cells is facilitated using a digital nanoplasmonic microarray immunosensor. (a) A schematic illustrates the design of this microfluidic nanoplasmonic microarray immunosensor. (b) The principle behind this immunosensor involves cytokine molecules forming a sandwich structure on immobilized capture antibodies (CAbs) with detection antibodies (DAbs). These structures are then labeled with gold nanoparticles (AuNPs) that produce a strong nanoplasmonic scattering signal [<a href="#B181-pharmaceutics-16-01228" class="html-bibr">181</a>]. Copyright 2023 © 2023 Elsevier B.V. Full article ">

15 pages, 2776 KiB

Open AccessArticle

Development of Biolayer Interferometry (BLI)-Based Double-Stranded RNA Detection Method with Application in mRNA-Based Therapeutics and Vaccines

by Dharia Sara Silas, Bindiya Juneja, Keerat Kaur, Muralikrishna Narayanareddy Gari, Yingjian You, Youmi Moon, Yizhuo Chen, Srishti Arora, Johanna Hansen, Kathir Muthusamy, Yue Fu, Nisha Palackal and Erica A. Pyles

Pharmaceutics 2024, 16(9), 1227; https://doi.org/10.3390/pharmaceutics16091227 - 19 Sep 2024

Viewed by 1915

Abstract

Background: In vitro-transcribed (IVT) mRNA has been established as a promising platform for therapeutics and vaccine development. Double-stranded RNA (dsRNA) is a major impurity of IVT mRNA and can trigger unfavored immune responses, potentially causing adverse events in patients. Existing dsRNA detection and [...] Read more.

Background: In vitro-transcribed (IVT) mRNA has been established as a promising platform for therapeutics and vaccine development. Double-stranded RNA (dsRNA) is a major impurity of IVT mRNA and can trigger unfavored immune responses, potentially causing adverse events in patients. Existing dsRNA detection and quantitation methods, such as gel electrophoresis, ELISA, or homogeneous time-resolved fluorescence (HTRF), have low sensitivity or are time-consuming. A recently published lateral flow immunoassay (LFSA) was shown to be fast, but it lacks the sensitivity for dsRNA with uridine modifications. Methods: In this study, we provided a possible explanation for the reduced sensitivity of existing quantitation methods for dsRNA with modified uridines by characterizing the binding affinities of commonly used anti-dsRNA antibodies. Then, a rapid and sensitive biolayer interferometry (BLI) dsRNA detection assay utilizing Flock House Virus (FHV) B2 protein was developed to overcome the challenges in dsRNA detection and the reduced sensitivity. Results: This assay allows the detection of dsRNA with different uridine modifications (ψ, m1ψ, 5 moU) with similar sensitivity as dsRNA without modification. Furthermore, we demonstrated this method can be used to quantify both short and long dsRNA, as well as hairpin-structured dsRNA, providing a more comprehensive detection for dsRNA impurities. Moreover, we applied this assay to monitor dsRNA removal through a purification process. Conclusions: Taken together, this BLI method could enable real-time monitoring of impurities in IVT mRNA production to prevent immunogenicity stemming from dsRNA. Full article

(This article belongs to the Special Issue State-of-Art in mRNA Therapeutics and Gene Delivery)

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Full article ">Figure 1
Determination of B2 binding affinity for dsRNA with different modifications. Binding sensorgrams and binding affinity determination of B2 for 700 bp dsRNA with different uridine modifications. (Top) (from left to right): representative binding sensorgrams of B2 with 700 bp-U, 700 bp-ψ, 700 bp-m1ψ or 700 bp-5 moU dsRNA. (Bottom): corresponding steady-state analysis using binding sensorgrams on the top (BLI signal plotted against B2 concentration) was utilized to determine B2 to dsRNA binding affinity (KD). All experiments were performed in triplicate. Full article ">Figure 2
Illustration of BLI dsRNA detection assay. BLI dsRNA detection assay with representative sensorgrams with dotted line separating each step. Step (1) establish baseline in assay buffer; (2) immobilize B2-Biotin on SA sensors; (3) wash to remove unbound B2-Biotin; (4) serially diluted dsRNA binding to B2 surface. Full article ">Figure 3
BLI dsRNA detection assay validation with HTRF assay. dsRNA detection by BLI dsRNA detection assay showed a similar trend as HTRF dsRNA assay for three IVT mRNA samples: (Left), BLI assay responses for mRNA-1 to mRNA-3 (with blank signal subtracted). (Right), HTRF signal (ratio) measured for mRNA-1 to mRNA-3 (with blank signal subtracted). All IVT mRNA samples were tested at 5 µg/mL in triplicates (BLI) and duplicates (HTRF). Full article ">Figure 4
BLI dsRNA detection assay specificity and interference testing. Testing specificity and interference using chemically similar nucleic acids on BLI dsRNA detection assay: (A) Specificity testing: BLI signal is shown for 700 bp-U, 700 bp-m1ψ, ssRNA (100-fold), dsDNA (100-fold), 142 bp dsRNA, and Poly(I:C). (B) Interference testing: BLI signal of 700 bp-U compared against 700 bp-U in the presence of 100-fold or 200-fold excess of ssRNA or dsDNA. (C) Interference testing: BLI signal of 700 bp-U was compared against 700 bp-m1ψ in the presence of 100-fold or 200-fold excess of ssRNA or dsDNA. All experiments were performed in triplicate. Full article ">Figure 5
Comparison of impact of uridine modifications on standard curves for BLI, HTRF, and ELISA method for dsRNA quantitation. Comparison of impact of uridine modifications on standard curves for BLI, HTRF, and ELISA methods. From top to bottom, overlay of 700 bp dsRNA standards with different modifications for (A) BLI dsRNA detection assay; (B) dsRNA quantitation HTRF assay; (C) dsRNA quantitation J2 ELISA. Full article ">Figure 6
Comparison of dsRNA standards of different lengths on BLI, HTRF, and ELISA methods. Comparison of dsRNA standards of different lengths using BLI, HTRF, and ELISA methods. From top to bottom, overlay of dsRNA standards of different lengths for (A) BLI dsRNA detection assay; (B) dsRNA quantitation HTRF assay; (C) dsRNA quantitation J2 ELISA. Full article ">Figure 7
BLI dsRNA assay can monitor dsRNA reduction through purification process. BLI dsRNA detection assay utilized to monitor dsRNA level through purification process. Two IVT mRNA transcripts (mRNA-4 and mRNA-5) were purified using ion pair reverse-phase (IPRP) chromatography. Before: IVT mRNA before IPRP purification. After: IVT mRNA purified by IPRP. (Top) panel: BLI response of mRNA-4 and mRNA-5 tested at 20 µg/mL in triplicates (with blank subtracted). (Bottom) panel: HTRF signal of mRNA-4 and mRNA-5 tested at 10 µg/mL in triplicates (with blank subtracted). Full article ">

15 pages, 4031 KiB

Open AccessArticle

Magnetic Nanoparticles with On-Site Azide and Alkyne Functionalized Polymer Coating in a Single Step through a Solvothermal Process

by Romualdo Mora-Cabello, David Fuentes-Ríos, Lidia Gago, Laura Cabeza, Ana Moscoso, Consolación Melguizo, José Prados, Francisco Sarabia and Juan Manuel López-Romero

Pharmaceutics 2024, 16(9), 1226; https://doi.org/10.3390/pharmaceutics16091226 - 19 Sep 2024

Viewed by 1130

Abstract

Background/Objectives: Magnetic Fe₃O₄ nanoparticles (MNPs) are becoming more important every day. We prepared MNPs in a simple one-step reaction by following the solvothermal method, assisted by azide and alkyne functionalized polyethylene glycol (PEG400) polymers, as well as by PEG6000 [...] Read more.

Background/Objectives: Magnetic Fe₃O₄ nanoparticles (MNPs) are becoming more important every day. We prepared MNPs in a simple one-step reaction by following the solvothermal method, assisted by azide and alkyne functionalized polyethylene glycol (PEG400) polymers, as well as by PEG6000 and the polyol β-cyclodextrin (βCD), which played a crucial role as electrostatic stabilizers, providing polymeric/polyol coatings around the magnetic cores. Methods: The composition, morphology, and magnetic properties of the nanospheres were analyzed using Transmission Electron and Atomic Force Microscopies (TEM, AFM), Nuclear Magnetic Resonance (NMR), X-ray Diffraction Diffractometry (XRD), Fourier-Transform Infrared Spectroscopy (FT-IR), Matrix-Assisted Laser Desorption/Ionization (MALDI) and Vibrating Sample Magnetometry (VSM). Results: The obtained nanoparticles (@Fe₃O₄-PEGs and @Fe₃O₄-βCD) showed diameters between 90 and 250 nm, depending on the polymer used and the Fe₃O₄·6H₂O precursor concentration, typically, 0.13 M at 200 °C and 24 h of reaction. MNPs exhibited superparamagnetism with high saturation mass magnetization at room temperature, reaching values of 59.9 emu/g (@Fe₃O₄-PEG6000), and no ferromagnetism. Likewise, they showed temperature elevation after applying an alternating magnetic field (AMF), obtaining Specific Absorption Rate (SAR) values of up to 51.87 ± 2.23 W/g for @Fe₃O₄-PEG6000. Additionally, the formed systems are susceptible to click chemistry, as was demonstrated in the case of the cannabidiol-propargyl derivative (CBD-Pro), which was synthesized and covalently attached to the azide functionalized surface of @Fe₃O₄-PEG400-N₃. Prepared MNPs are highly dispersible in water, PBS, and citrate buffer, remaining in suspension for over 2 weeks, and non-toxic in the T84 human colon cancer cell line, Conclusions: indicating that they are ideal candidates for biomedical applications. Full article

(This article belongs to the Special Issue Recent Advances in Biomedical Applications of Magnetic Nanomaterials)

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The microscopy study results: (A) TEM image of @Fe3O4-PEG400 nanospheres, (B) TEM image of a single @Fe3O4-PEG400 nanosphere, (C) AFM image of @Fe3O4-PEG400, and (D) AFM image of @Fe3O4-PEG400 single nanosphere. Full article ">Figure 2
TEM images of: (A) @Fe3O4-PEG400-Pro, (B) @Fe3O4-PEG400-N3, (C) @Fe3O4-βCD, and (D) @Fe3O4-PEG6000. Full article ">Figure 3
Representative XRD diffractogram of Fe3O4 nanoparticles coated with PEG (@Fe3O4-PEG6000). Full article ">Figure 4
FT-IR Spectra of: (A) @Fe3O4-PEG400-Pro, (B) @Fe3O4-PEG400-N3, (C) @Fe3O4-βCD, and (D) @Fe3O4-PEG6000 samples. Full article ">Figure 5
Magnetization curves of @Fe3O4 coated with βCD, PEG400, or PEG6000. Full article ">Figure 6
Hyperthermia and in vitro proliferation analyses: (A) Temperature rise obtained after application of an alternating magnetic field (385 kHz; 28 kA/m) during 25 min for MNPs coated with PEG400, PEG6000, and βCD at a concentration of 0.5 mg/mL of Fe. The data were represented as the mean of 3 measurements ± standard deviation; (B) In vitro proliferation assay of MNPs coated with PEG400, PEG6000, and βCD at 72 h of exposition. Graphs show the percentage of proliferation of T84 at doses ranging from 1–100 µg/mL of Fe3O4. Results were expressed as mean ± SD of triplicate cultures. Full article ">Scheme 1
Two-step synthesis of PEG400-N3 and synthesis of PEG400-Pro. Full article ">Scheme 2
Synthesis of propargyl derivatives of CBD and preparation of @Fe3O4-PEG400-CBD. Full article ">

24 pages, 65791 KiB

Open AccessArticle

Antimicrobial Hydroxyethyl-Cellulose-Based Composite Films with Zinc Oxide and Mesoporous Silica Loaded with Cinnamon Essential Oil

by Ludmila Motelica, Denisa Ficai, Gabriela Petrisor, Ovidiu-Cristian Oprea, Roxana-Doina Trușcǎ, Anton Ficai, Ecaterina Andronescu, Ariana Hudita and Alina Maria Holban

Pharmaceutics 2024, 16(9), 1225; https://doi.org/10.3390/pharmaceutics16091225 - 19 Sep 2024

Cited by 4 | Viewed by 1538

Abstract

Background: Cellulose derivatives are gaining much attention in medical research due to their excellent properties such as biocompatibility, hydrophilicity, non-toxicity, sustainability, and low cost. Unfortunately, cellulose does not exhibit antimicrobial activity. However, derivatives like hydroxyethyl cellulose represent a proper matrix to incorporate [...] Read more.

Background: Cellulose derivatives are gaining much attention in medical research due to their excellent properties such as biocompatibility, hydrophilicity, non-toxicity, sustainability, and low cost. Unfortunately, cellulose does not exhibit antimicrobial activity. However, derivatives like hydroxyethyl cellulose represent a proper matrix to incorporate antimicrobial agents with beneficial therapeutic effects. Methods: Combining more antimicrobial agents into a single composite material can induce stronger antibacterial activity by synergism. Results: Therefore, we have obtained a hydroxyethyl-cellulose-based material loaded with zinc oxide nanoparticles and cinnamon essential oil as the antimicrobial agents. The cinnamon essential oil was loaded in mesoporous silica particles to control its release. Conclusions: The composite films demonstrated high antibacterial activity against Staphylococcus aureus and Escherichia coli strains, impairing the bacterial cells’ viability and biofilm development. Such antimicrobial films can be used in various biomedical applications such as topical dressings or as packaging for the food industry. Full article

(This article belongs to the Special Issue The 15th Anniversary of Pharmaceutics—Biotechnological Drugs: Innovations in Preparation and Delivery Strategies)

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17 pages, 2738 KiB

Open AccessArticle

Deciphering Chemical Rules for Drug Penetration into Strongyloides

by Miguel Marín, Javier Sánchez-Montejo, Sergio Ramos, Antonio Muro, Julio López-Abán and Rafael Peláez

Pharmaceutics 2024, 16(9), 1224; https://doi.org/10.3390/pharmaceutics16091224 - 19 Sep 2024

Viewed by 787

Abstract

Background: Strongyloidiasis, a parasitic infection, presents a significant public health challenge in tropical regions due to the limited repertoire of effective treatments. The screening of chemical libraries against the therapeutically relevant third-stage larvae (L3) of the model parasite Strongyloides venezuelensis yielded meager success [...] Read more.

Background: Strongyloidiasis, a parasitic infection, presents a significant public health challenge in tropical regions due to the limited repertoire of effective treatments. The screening of chemical libraries against the therapeutically relevant third-stage larvae (L3) of the model parasite Strongyloides venezuelensis yielded meager success rates. This situation is reminiscent of Gram-negative bacteria, where drug entry is a limiting factor. Methods: Here, we set out to determine whether similar barriers are in place and establish whether structural and property requirements exist for anti-strongyloides drug discovery. We focused on dyes as their uptake and effects on viability can be independently assessed in the multicellular parasite, thus providing a means to study the possibility of similar entry rules. We tested different dyes in in vitro assays on L3s. Results: We found that staining was necessary to reduce parasite viability, with some dyes achieving anti-strongyloides effects at concentrations similar to those of the reference drug, ivermectin (IV). Some dyes also showed activity against female adults at concentrations well below that of ivermectin. Unfortunately, the most potent dye, Methylene Blue, was unable to prevent the infection in a preliminary in vivo mouse model assay, presumably due to fast dye clearance. Structural analysis showed that positive charges facilitated the access of the compounds to the L3 tissue, thus providing a structural tool for the introduction of activity. For female adults, low globularity is additionally required. As a proof of concept, we added a positive charge to an inactive compound of one of our chemical libraries and re-determined the activity. Conclusions: These findings allow us to establish structural rules for parasite entry that could be of interest for future drug screening or drug development campaigns. These rules might also be applicable to other related parasites. Full article

(This article belongs to the Special Issue Novel Pharmaceuticals Development and Delivery Systems for the Treatment of Parasitic Diseases, 2nd Edition)

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Dose–response curves and calculation of IC50 values for Methylene Blue, Fuchsine, and Crystal Violet after 72 h of treatment. Full article ">Figure 2
Images of L3s stained with Methylene Blue (100×) (A), Fuchsine (400×) (B), and Crystal Violet (400×) (C). L3s were treated with each compound at 10 µM and photographed after 24 h. Full article ">Figure 3
Stained intestine inside an adult of S. venezuelensis treated with Methylene Blue at 10 µM for 72 h (A). Stained S. venezuelensis adults (red arrow) and eggs (blue arrows) treated with Methylene Blue at 10 µM for 72 h (B). Stained intestine in adults treated with Brilliant Cresyl Blue at 10 µM for 72 h (C). Adults treated with propidium iodide at 10 µM for 72 h, showing diffuse and intense points in inner structures ((D) white field and (E) fluorescence). Full article ">Figure 4
Eggs per gram of feces through infection and number of females recovered at day 8 p.i. in mice infected with 3000 L3s and treated with Methylene Blue administered by oral gavage and Methylene Blue and Crystal Violet provided ad libitum in drinking water. Full article ">Scheme 1
In vivo procedure. Mice were infected at day 0 and treated at days 0, 1, 2, 3, 4, and 5 p.i. Feces were recovered at days 4, 5, 6, 7, and 8 p.i. Full article ">

17 pages, 1508 KiB

Open AccessArticle

Diagnosis of Prostate Cancer with a Neurotensin–Bombesin Radioligand Combination—First Preclinical Results

by Maria Bibika, Panagiotis Kanellopoulos, Maritina Rouchota, George Loudos, Berthold A. Nock, Eric P. Krenning and Theodosia Maina

Pharmaceutics 2024, 16(9), 1223; https://doi.org/10.3390/pharmaceutics16091223 - 19 Sep 2024

Viewed by 861

Abstract

Background: The concept of radiotheranostics relies on the overexpression of a biomolecular target on malignant cells to direct diagnostic/therapeutic radionuclide-carriers specifically to cancer lesions. The concomitant expression of more than one target in pathological lesions may be elegantly exploited to improve diagnostic sensitivity [...] Read more.

Background: The concept of radiotheranostics relies on the overexpression of a biomolecular target on malignant cells to direct diagnostic/therapeutic radionuclide-carriers specifically to cancer lesions. The concomitant expression of more than one target in pathological lesions may be elegantly exploited to improve diagnostic sensitivity and therapeutic efficacy. Toward this goal, we explored a first example of a combined application of [^99mTc]Tc-DT11 (DT11, N₄-Lys(MPBA-PEG4)-Arg-Arg-Pro-Tyr-Ile-Leu-OH; NTS₁R-specific) and [^99mTc]Tc-DB7(DB7, N₄-PEG2-DPhe-Gln-Trp-Ala-Val-Gly-His-Leu-NHEt; GRPR-specific) in prostate cancer models. Methods: Accordingly, the behavior of [^99mTc]Tc-DT11 was compared with that of the [^99mTc]Tc-DT11+[^99mTc]Tc-DB7 mixture in prostate adenocarcinoma PC-3 cells and xenografts in mice. The impact of stabilizing both radiotracers by Entresto^®, as a source of the potent neprilysin inhibitor sacubitrilat, was also investigated. Results: The PC-3 cell binding of the [^99mTc]Tc-DT11+[^99mTc]Tc-DB7 mixture surpassed that of [^99mTc]Tc-DT11. Likewise, the PC-3 tumor uptake of the [^99mTc]Tc-DT11+[^99mTc]Tc-DB7 mixture at 4 h post-injection was superior (7.70 ± 0.89%IA/g) compared with [^99mTc]Tc-DT11 (4.23 ± 0.58%IA/g; p < 0.0001). Treatment with Entresto^® led to further enhancement of the tumor uptake (to 11.57 ± 1.92%IA/g; p < 0.0001). Conclusions: In conclusion, this first preclinical study on prostate cancer models revealed clear advantages of dual NTS₁R/GRPR targeting, justifying further assessment of this promising concept in other cancer models. Full article

(This article belongs to the Special Issue Peptide–Drug Conjugates for Targeted Delivery)

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Chemical structures of (a) [99mTc]Tc-DT11 (DT11, N4-Lys(MPBA-PEG4)-Arg-Arg-Pro-Tyr-Ile-Leu-OH; N4, 6-(carboxy)-1,4,8,11-tetraazaundecane; MPBA = 4-(4-methylphenyl)butyric acid; PEG4: 14-amino-3,6,9,12-tetraoxatetradecan-1-oic acid) and (b) [99mTc]Tc-DB7 (DB7, N4-PEG2-DPhe-Gln-Trp-Ala-Val-Gly-His-Leu-NHEt; PEG2, 8-Amino-3,6-dioxaoctanoic acid). Full article ">Figure 2
Percentage of receptor-specific radioligand uptake after 1 h incubation at 37 °C in PC-3 cells, comprising internalized (solid bars on the bottom) and membrane-bound fractions (checkered bars on top) corresponding to [99mTc]Tc-DT11 (NTS1R-specific: light blue bars on the left), (GRPR-specific: light pink bars in the middle, [<a href="#B36-pharmaceutics-16-01223" class="html-bibr">36</a>]), and [99mTc]Tc-DT11+[99mTc]Tc-DB7 (NTS1R+GRPR-specific: violet bars on the right); results represent mean values ± sd, n = 3 (each in triplicate) statistically significant differences are denoted with ****, correspond to p < 0.0001. Full article ">Figure 3
Selected biodistribution data of [99mTc]Tc-DT11 (average %IA/g ± sd at 4 h pi) in PC-3 xenograft-bearing SCID mice; for comparison purposes, bars from left to right in each set correspond to block (animals co-injected excess NT (100 µg), n = 3; first bar: gray with square filling), controls (second bar: light blue, n = 4) and Entresto® (mice orally receiving Entresto® 30 min before injection of the radioligand, n = 4; third bar: dark blue). Values are shown for kidneys, intestines, pancreases and PC-3 tumors; statistically significant differences are denoted: **, for p < 0.01 and ****, for p < 0.0001. Full article ">Figure 4
Selected biodistribution data (average %IA/g ± sd; n = 4) in SCID mice bearing PC-3 xenografts at 4 h pi of an equimolar [99mTc]Tc-DT11+[99mTc]Tc-DB7 mixture for easy comparison purposes for kidneys, intestines, pancreases and PC-3 tumors. Bars from left to right in each set correspond to controls (first bar: light violet), Entresto® (mice having orally received Entresto® 30 min prior to the injection of the [99mTc]Tc-DT11+[99mTc]Tc-DB7 mixture; second bar: dark violet), block 1 (animals co-injected with 100 µg NT; third bar: pink checkered filling), block 2 (animals co-injected with 50 µg [Tyr4]BBN; fourth bar: blue checkered filling) and block 3 (animals co-injected with 100 µg NT and 50 µg [Tyr4]BBN; fifth bar: violet checkered filling). Statistically significant differences are denoted: ****, for p < 0.0001. Full article ">Figure 5
Static whole body SPECT/CT images 4 h pi of an equimolar [99mTc]Tc-DT11+ [99mTc]Tc-DB7 mixture alone (a,b) or together with 100 µg NT and 50 µg [Tyr4]BBN for twin NTS1R and GRPR-blockade (c). All animals had orally received Entresto® 30 min in advance. Yellow arrows indicate the position of PC-3 xenografts; images correspond to maximum intensity projections, and intensity of uptake is represented by the color scale on the right of each image. Blue corresponds to the lowest and yellow to the highest value. Full article ">

24 pages, 3085 KiB

Open AccessArticle

Comprehensive Analysis of Cetuximab Critical Quality Attributes: Impact of Handling on Antigen-Antibody Binding

by Alicia Torres-García, Anabel Torrente-López, Jesús Hermosilla, Amparo Hernández, Antonio Salmerón-García, José Cabeza and Natalia Navas

Pharmaceutics 2024, 16(9), 1222; https://doi.org/10.3390/pharmaceutics16091222 - 19 Sep 2024

Viewed by 1108

Abstract

Background/Objectives: Cetuximab, formulated in Erbitux^® (5 mg/mL), is a therapeutic monoclonal antibody (mAb) widely used in several cancer treatments. Currently, there is insufficient knowledge about the behavior of cetuximab with regard to the risk associated with its routine handling or unintentional mishandling [...] Read more.

Background/Objectives: Cetuximab, formulated in Erbitux^® (5 mg/mL), is a therapeutic monoclonal antibody (mAb) widely used in several cancer treatments. Currently, there is insufficient knowledge about the behavior of cetuximab with regard to the risk associated with its routine handling or unintentional mishandling in hospitals. Forced degradation studies can simulate these conditions and provide insights into the biophysical and biochemical properties of mAbs. Methods: In this study, we conducted a deep physicochemical and functional characterization of the critical quality attributes of cetuximab in control samples and under controlled degraded conditions, including freeze–thaw cycles, heat, agitation, and light exposure. To achieve this purpose, we used a set of proper analytical techniques, including CD, IT-FS, DLS, SE/UHPLC-UV, UHPLC-MS/MS, and ELISA, to check functionality based on antigen–antibody binding. Results: The results revealed that light exposure was the stress stimuli with the greatest impact on the drug product, leading to the formation of non-natural oligomers, fragmentation, and oxidation of methionine residues. Additionally, cetuximab (Erbitux^®, 5 mg/mL) showed a tendency to aggregate when submitted to 60 °C for 1 h. In terms of functionality, cetuximab (Erbitux^®, 5 mg/mL) samples were found to be affected when subjected to freeze–thaw cycles, 60 °C (1 h), and when exposed to light (daylight with room temperature excursion and accelerated light exposure). Conclusions: Thus, we suggest that Erbitux^® (5 mg/mL) should be shielded from these environmental conditions, as they compromise both the safety and efficacy of the drug product. Full article

(This article belongs to the Special Issue Physical and Chemical Stability of Drug Formulation)

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Far-UV CD spectra of cetuximab (5 mg/mL) stressed and control samples. Full article ">Figure 2
Intrinsic tryptophan fluorescence emission spectra of cetuximab (Erbitux®, 5 mg/mL) stressed and control samples. Full article ">Figure 3
Representative chromatograms SE/UHPLC-UV cetuximab samples. (A) Freeze-thaw cycles stress, (B) agitation stress, (C) temperature stress, and (D) light stress. The retention times indicated in the chromatograms are the means of three replicates. Full article ">Figure 4
Cetuximab primary sequence. Regions highlighted in green, orange, and blue correspond to CDR1, CDR2, and CDR3, respectively. Adapted from Ayoub D et al. [<a href="#B34-pharmaceutics-16-01222" class="html-bibr">34</a>]. Full article ">Figure 5
Relative abundance of most important PTMs identified in cetuximab. (A) Oxidations, (B) pyroglutamination, (C) glycosylation located on H-N88 residue, and (D) glycosylation located on H-N299 residue. Error bars depict standard deviation (n = 3). Full article ">Figure 6
Comprehensive glycan profile of cetuximab. The various isoforms are arranged in decreasing order of relative abundance (%) based on the control sample both in (A) H-N88 residue and (B) H-N299 residue. Full article ">Figure 7
Calibration curve for cetuximab ELISA analysis. (A) Standard curve. (B) Logarithmic scale curve. Full article ">Figure 8
ELISA binding assay comparing control and stressed samples. (A) Freeze-thaw cycles stress, (B) agitation stress, (C) temperature stress, and (D) light stress. Conditions marked with asterisk (*) showed statistical differences when compared to the control. Full article ">

15 pages, 1565 KiB

Open AccessArticle

Aerosol of Enoximone/Hydroxypropyl-β-Cyclodextrin Inclusion Complex, Biopharmaceutical Evidence for ARDS Applicability

by Chiara Migone, Brunella Grassiri, Lucia Vizzoni, Angela Fabiano, Baldassare Ferro, Ylenia Zambito and Anna Maria Piras

Pharmaceutics 2024, 16(9), 1221; https://doi.org/10.3390/pharmaceutics16091221 - 19 Sep 2024

Viewed by 817

Abstract

Background: Phosphodiesterase (PDE) inhibitors are gaining interest in the context of pulmonary pathologies. In particular, the PDE3 inhibitor enoximone (ENXM) has shown potential relative to the cure of asthma, chronic obstructive pulmonary disease (COPD), and acute respiratory distress syndrome (ARDS). Despite its administration [...] Read more.

Background: Phosphodiesterase (PDE) inhibitors are gaining interest in the context of pulmonary pathologies. In particular, the PDE3 inhibitor enoximone (ENXM) has shown potential relative to the cure of asthma, chronic obstructive pulmonary disease (COPD), and acute respiratory distress syndrome (ARDS). Despite its administration via inhalation being planned for use against COVID-19 related ARDS (C-ARDS), presently, no inhalable medicine containing ENXM is available. Objectives: This study aims to develop a new formulation suitable for pulmonary administration of ENXM. Methods: A solution for nebulization, based on the complex between ENXM and Hydroxypropyl-β-Cyclodextrin (HPβCD) (ENXM/HPβCD) is developed. The obtained solution is characterized in terms of aerodynamic distributions and biopharmaceutical features. Results: The evaluation of the aerosol droplets indicates a good bronchi–lung distribution of the drug. Biological evaluations of the air–liquid interface (ALI) in an in vitro lung cell model demonstrates that ENXM/HPβCD is capable of a local direct effect, increasing intracellular cyclic adenosine monophosphate (cAMP) levels and protecting from oxidative stress. Conclusions: This study offers a promising advance in the optimization of enoximone delivery to the lungs. Full article

(This article belongs to the Special Issue Inhalable Drugs for the Treatment of Chronic Respiratory Diseases)

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Job’s plot representation of the ENXM/HPβCD complex. ΔA stands for the difference between absorbance values. The ENXM molar fraction value (R) at the maximum of the curve is 0.5. Full article ">Figure 2
In vitro cytotoxicity of ENXM/HPβCD and its components on NCI-H441 cells. Data expressed as viability percentage for (a) ENXM concentration and (b) HPβCD concentration (n = 6). Full article ">Figure 3
Maintenance of ALI monolayer integrity upon droplet impact under nebulization in the TSI setup. NSS was aerosolized directly onto the NCI-H441 cell monolayer. Transwell® insert NCI-H441 cell monolayers were placed at the base of the lower chamber of a TSI. The nebulization was performed under different duration times. TEER values before and after nebulization and micrographs of NCI-H441 cell monolayers fixed and stained post-nebulization (10× magnification, same scale bar for all micrographs) are presented. Full article ">Figure 4
In vitro evaluations of drug activity, drug dose 1.5 μg per ALI monolayer. (a) Direct effect of the drug, acting as PDE-3i, thus increasing the intracellular cAMP levels: non-treated cells, drug saline suspension (plain ENXM), plain ENXM administered after NSS nebulization for 30 s (impacting NSS + plain ENMX), HPβCD (0.25 mg), and ENXM/HPβCD were nebulized for 30 s. *** p < 0.001 vs. all the other samples. Data were normalized on total protein concentration and expressed as pmol of cAMP per μg of protein. (b) Cell viability of H2O2 stressed cells pretreated with the drug: plain ENXM administered after NSS nebulization for 30 s and ENXM/HPβCD nebulized for 30 s. *** p < 0.001 vs. all the other samples. Full article ">Figure 5
Permeation of the drug across the ALI monolayer of the NCI-H441 cell line, after simulating in vitro the impact of the drug on the deep lung. The cell monolayer was placed in the second stage of a TSI during sample nebulization. Full article ">

22 pages, 3597 KiB

Open AccessArticle

Enhanced Antitumor Efficacy of Cytarabine and Idarubicin in Acute Myeloid Leukemia Using Liposomal Formulation: In Vitro and In Vivo Studies

by Chunxia Zhu, Yang Liu, Xiaojun Ji, Yaxuan Si, Xianhao Tao, Xiaohua Zhang and Lifang Yin

Pharmaceutics 2024, 16(9), 1220; https://doi.org/10.3390/pharmaceutics16091220 - 19 Sep 2024

Cited by 1 | Viewed by 951

Abstract

Background: Acute myeloid leukemia (AML) is the most common type of acute leukemia among adults with the recommend therapy of combination of cytarabine and idarubicin in the induction phase. The uncoordinated pharmacokinetics prevent adequate control of drug ratio following systemic administration. Therefore, the [...] Read more.

Background: Acute myeloid leukemia (AML) is the most common type of acute leukemia among adults with the recommend therapy of combination of cytarabine and idarubicin in the induction phase. The uncoordinated pharmacokinetics prevent adequate control of drug ratio following systemic administration. Therefore, the dual-loaded liposomes containing cytarabine and idarubicin for synergistic effects were proposed and investigated. Methods: The molar ratio of cytarabine and idarubicin for synergistic effects was investigated. The dual-loaded liposomes were prepared and characterized by particle size, zeta potential, encapsulation efficiency, cryo-Transmission electron microscopy (cryo-TEM), and in vitro stability. The in vitro cytotoxicity and cell uptake of liposomes were determined within CCRF-CEM cells. The PK experiments was carried out in male SD rats. The in vivo antitumor effect was carried out within CD-1 nude female mice. The antitumor mechanism of liposomes was investigated. Results: The synergistic molar ratios were found to be in the range of 20:1~40:1. The size distribution of the dual-loaded liposomes was approximately 100 nm with PDI ≤ 0.1, a zeta potential of approximately −30 mV, an entrapment efficiency of cytarabine and idarubicin of >95% with spherical structure and uniform distribution, and in vitro stability for 21 d. The drugs in the liposomes can be quickly uptaken by the leukemia cells. The PK experiments showed that the molar ratio of cytarabine to idarubicin in plasma was maintained at 30:1 within 4 h. The efficacy of liposomes was significantly enhanced. Conclusions: The dual-loaded liposomes containing cytarabine and idarubicin showed enhanced antitumor efficacy. Full article

(This article belongs to the Special Issue Innovative Drug Delivery Systems for Enhanced Therapeutic Efficacy in Cancers, Liver Disease and Psoriasis)

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The proportion of cells with different molar ratios of cytarabine and idarubicin exerting synergistic effects. (A) When Fa values were 0.5, 0.75, and 0.9, the ratio of cytarabine to idarubicin was set at 5:1, 10:1, 20:1, 30:1, 40:1, and 50:1, the proportion of all 13 cell lines showing synergistic effect (CI value less than 1). (B) When Fa values were 0.5, 0.75, and 0.9, the ratio of cytarabine to idarubicin was set at 5:1, 10:1, 20:1, 30:1, 40:1, and 50:1, the proportion of nine leukemia cells showing synergistic effect (CI value less than 1). Full article ">Figure 2
Typical profile of the dual drug liposome with a cytarabine/idarubicin mole ratio of 30:1 with Glu-Cu2+ as a carrier ion. (A): Diagram of intensity-particle size distribution. The peak indicates the particle size distribution, the curve indicates the cumulative percentage. (B): Diagram of Cyro-TEM. Full article ">Figure 3
In vitro cell uptake of cytarabine-idarubicin liposome on CCRF-CEM cells. (A) The proliferation inhibition curve of CCRF-CEM cells treated with Lipo-5; Lipo-5 was diluted three times from 30,000 nM to 4.6 nM (measured by cytarabine concentration), and the experiment was repeated three times. (B) The proliferation inhibition curve of CCRF-CEM cells treated with combined free drugs of cytarabine-idarubicin (molar ratio at 30:1); free drugs were diluted three times from 30,000 nM to 4.6 nM (measured by cytarabine concentration), and the experiment was repeated three times. Full article ">Figure 4
In vitro cell uptake of cytarabine-idarubicin liposome on CCRF-CEM cells. The cell membrane was stained with DiD for 15 min, and then the dyestuff was removed by centrifugation, and CCRF-CEM cells were incubated with Lipo-1 (500 μM) for 0.5 h, 2 h, 4 h, 8 h, and 16 h. After incubation, nuclei were stained with Hoechst for 0.5 h, and then cells were centrifuged to remove dyestuff. After that, cells were washed three times with cold HBSS and visualized using a high-content screening (HCS). Full article ">Figure 5
PK characteristics of CD-1 mice treated with cytarabine-idarubicin liposome. CD-1 mice were intravenously injected with three kinds of prescription cytarabine-idarubicin liposomes (the molar ratio of cytarabine to idarubicin was 10:1, the dose of cytarabine was 12 mg/kg, and the dose of idarubicin was 2.63 mg/kg) or the combination of cytarabine-idarubicin free drugs (the dose of cytarabine was 600 mg/kg, and the dose of idarubicin was 1.8 mg/kg). The plasma and bone marrow of mice were collected at different time points to detect the concentrations of cytarabine and idarubicin; three samples were collected at each time point. (A) Drug concentration-time curves of cytarabine and idarubicin within plasma after injection of Lipo-1 and combined free drugs in CD-1 mice. (B) Drug concentration-time curves of cytarabine and idarubicin within bone marrow after injection of Lipo-1 and combined free drugs in CD-1 mice. (C) Drug concentration-time curves of cytarabine and idarubicin within plasma after injection of Lipo-2 and combined free drugs in CD-1 mice. (D) Drug concentration-time curves of cytarabine and idarubicin within bone marrow after injection of Lipo-2 and combined free drugs in CD-1 mice. (E) Drug concentration-time curves of cytarabine and idarubicin within plasma after injection of Lipo-3 and combined free drugs in CD-1 mice. (F) Drug concentration-time curves of cytarabine and idarubicin within bone marrow after injection of Lipo-3 and combined free drugs in CD-1 mice. (G) The molar ratio changes of cytarabine to idarubicin in plasma at different time points in CD-1 mice after intravenous administration of Lipo-1, Lipo-2, and Lipo-3. (H) The molar ratio changes of cytarabine to idarubicin in plasma at different time points in CD-1 mice after intravenous administration of combined free drugs. (I) The molar ratio changes of cytarabine to idarubicin in bone marrow at different time points in CD-1 mice after intravenous administration of Lipo-1, Lipo-2, and Lipo-3. (J) The molar ratio changes of cytarabine to idarubicin in bone marrow at different time points in CD-1 mice after intravenous administration of combined free drugs. Full article ">Figure 6
Efficacy of cytarabine-idarubicin liposomes in WEHI-3B ascites model. Female CD-1 nude mice were intravenously injected with Lipo-4 as 5 mg/kg, 10 mg/kg, 12 mg/kg, 15 mg/kg (as measured by cytarabine), free drug combinations as 200:0.6 mg/kg, 200:1.0 mg/kg, 200:1.5 mg/kg, 300:0.9 mg/kg, and 300:1.5 mg/kg on D1, D4, and D7 to explore the maximum tolerated dose of the liposome and free drug combination. (A) Weight change curves of CD-1 nude mice within 14 days after the last dose, with five animals per group. (B) Percentage change in body weight from baseline within 14 days after the last dose, with five animals per group. Female CD-1 nude mice were intravenously injected with Lipo-4, Lipo-5, Lipo-6, and free drug combinations on D1, D4, and D7 after the WEHI-3B ascites model was set successfully. (C) The weight change curve of each group of mice after modeling, with six animals per group. (D) Survival rate curves of mice in each group after modeling, with six animals per group. Full article ">Figure 7
Cell cycle analysis of Kasumi-1 cells treated with combined free drugs. Kasumi-1 cells were treated with combined free drugs to explore their effects on the cell cycle. (A) The proportion of cells blocked on the G0/G1 phase. (B) The proportion of cells blocked on the S phase. (C,D) cell cycle analysis as molar ratio was 5:1, (E,F) cell cycle analysis as molar ratio was 10:1, (G,H) cell cycle analysis as molar ratio was 20:1, (I,J) cell cycle analysis as molar ratio was 30:1, (K,L) cell cycle analysis as molar ratio was 40:1, (M,N) cell cycle analysis as molar ratio was 50:1. Data were shown as mean ± SD, * p < 0.05, ** p < 0.01, compared with the control group (A,B). The experiment was repeated four times. Full article ">Figure 8
Gene expression was associated with the cell cycle in Kasumi-1 cells as the concentration of cytarabine was 3.33 μM. Kasumi-1 cells were treated with combined free drugs to explore its effects on gene expression associated with the cycle cell cycle. Gene expression of CDK1 (A), CDK2 (B), CDK4 (C), CDK6 (D), CCNA2 (E), CCNB1 (F), CCND1 (G), CCND2 (H), CCND3 (I) and CCNE1 (J) was measured by qRT-PCR. Data were shown as mean ± SD, * p < 0.05, ** p < 0.01, *** p < 0.001, compared with the control group. The experiment was repeated three times. Full article ">

12 pages, 2394 KiB

Open AccessArticle

Plasmid DNA Delivery into the Skin via Electroporation with a Depot-Type Electrode

by Yuya Yoshida, Manami Aoki, Kalin Nagase, Koichi Marubashi, Hiroyuki Kojima, Shoko Itakura, Syuuhei Komatsu, Kenji Sugibayashi and Hiroaki Todo

Pharmaceutics 2024, 16(9), 1219; https://doi.org/10.3390/pharmaceutics16091219 - 18 Sep 2024

Viewed by 974

Abstract

Objectives: Non-viral mediated plasmid DNA transfection by electroporation (EP) is an established method for gene transfection. In this study, the usefulness of direct EP at an intradermal (i.d.) site (D_EP) with implanted electrodes to achieve a high protein [...] Read more.

Objectives: Non-viral mediated plasmid DNA transfection by electroporation (EP) is an established method for gene transfection. In this study, the usefulness of direct EP at an intradermal (i.d.) site (D_EP) with implanted electrodes to achieve a high protein expression level was investigated. In addition, D_EP application with various intervals with a low application voltage was also evaluated to confirm its effect on protein expression. Methods: Green fluorescent protein (GFP)- and luciferase-encoding DNA were administrated, and GFP and luciferase were evaluated. Results: A higher protein expression level was observed after green fluorescent protein (GFP)- and luciferase-encoding DNA were delivered by i.d. injection followed by D_EP application. When luciferase expression was observed with an in vivo imaging system, continuous expression was confirmed over 21 days after i.d. injection followed by D_EP at 100 V. This approach provided increased gene expression levels compared with conventional EP methods via the stratum corneum layer. In addition, the effect of application voltage on luciferase expression was investigated; two-time applications (repeated D_EP) at 20 V with 5 min intervals showed similar luciferase expression level to single D_EP application with 100 V. Histological observations showed the skin became thicker after a single D_EP at 100 V, whereas no apparent thickness changes were confirmed after repeated D_EP at 20 V with 5 min intervals. Conclusions: This study revealed that direct i.d. voltage application achieved high protein expression levels even at low voltages. Skin is a promising administration site for DNA vaccines, so this approach may be effective for DNA vaccine delivery into skin tissue. Full article

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

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15 pages, 10180 KiB

Open AccessArticle

Functionalized PLGA-Based Nanoparticles with Anti-HSV-2 Human Monoclonal Antibody: A Proof of Concept for Early Diagnosis and Targeted Therapy

by Melinda Mariotti, Noah Giacon, Ettore Lo Cascio, Margherita Cacaci, Simona Picchietti, Maura Di Vito, Maurizio Sanguinetti, Alessandro Arcovito and Francesca Bugli

Pharmaceutics 2024, 16(9), 1218; https://doi.org/10.3390/pharmaceutics16091218 - 18 Sep 2024

Viewed by 865

Abstract

Background: Functionalized nanoparticles (NPs) represent a cutting edge in innovative clinical approaches, allowing for the delivery of selected compounds with higher specificity in a wider time frame. They also hold promise for novel theranostic applications that integrate both diagnostic and therapeutic functions. Pathogens [...] Read more.

Background: Functionalized nanoparticles (NPs) represent a cutting edge in innovative clinical approaches, allowing for the delivery of selected compounds with higher specificity in a wider time frame. They also hold promise for novel theranostic applications that integrate both diagnostic and therapeutic functions. Pathogens are continuously evolving to try to escape the strategies designed to treat them. Objectives: In this work, we describe the development of a biotechnological device, Nano-Immuno-Probes (NIPs), for early detection and infections treatment. Human Herpes Simplex Virus 2 was chosen as model pathogen. Methods: NIPs consist of PLGA-PEG-Sulfone polymeric NPs conjugated to recombinant Fab antibody fragments targeting the viral glycoprotein G2. NIPs synthesis involved multiple steps and was validated through several techniques. Results: DLS analysis indicated an expected size increase with a good polydispersity index. Z-average and z-potential values were measured for PLGA-PEG-Bis-Sulfone NPs (86.6 ± 10.9 nm; –0.7 ± 0.3 mV) and NIPs (151 ± 10.4 nm; −5.1 ± 1.9 mV). SPR assays confirmed NIPs’ specificity for the glycoprotein G2, with an apparent K_D of 1.03 ± 0.61 µM. NIPs exhibited no cytotoxic effects on VERO cells at 24 and 48 h. Conclusions: This in vitro study showed that NIPs effectively target HSV-2, suggesting the potential use of these nanodevices to deliver both contrast agents as well as therapeutic compounds. Full article

(This article belongs to the Section Nanomedicine and Nanotechnology)

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Graphical abstract
Full article ">Figure 1
Panel (a) illustrates the sequence of steps involved in synthesizing and characterizing NIPs. The process begins with the synthesis of the polymer (Step 1), followed by the synthesis of nanoparticles via nanoprecipitation (Step 2). Once formed, the nanoparticles are functionalized with the antibody (Step 3), and finally, the particles undergo characterization (Step 4). Panel (b) depicts a schematic of the nanoprecipitation technique. In this technique, PLGA-PEG-Bis-sulfone polymer is dissolved in THF (tetrahydrofuran). This solution is then added dropwise to continuously stirred water. The mixture is left stirring until nanoparticles are formed. Full article ">Figure 2
Comparative evaluation on the agarose gel of the HC gene amplification. Amplicons were generated by PCR using the CG1z4HIS primer for the Histidine tag addition (lanes 1–2), or the CG1z unmodified primer (lanes 3–4) in combination with a mix of A (1–3) or F (2–4) variable primers. The anti-HSV-2 original plasmid was used as a template. Full article ">Figure 3
Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE; Coomassie blue staining) analysis: evaluation of dialysis efficiency. From left to right: protein molecular mass marker, anti-HSV-2 Fab pre-dialysis, anti-HSV-2 Fab post dialysis. Full article ">Figure 4
Negative staining (a,b) and representative immunogold images (c,d) of NPs and NIPs. (a) NPs showing spherical shape, size heterogeneity and smooth membranes; (b) Smooth membrane of spherical NIPs; (c) Detection of Fab epitopes on the surface of NIPs using gold conjugated Fab-specific antibody (dark particles); (d) NPs without staining on their surface in negative control. Bars: (a–d) 200 nm. Magnification rate: 100 K; scale bar: 200 nm. Full article ">Figure 5
Immunoblot of the PES membranes that flowed through the solution (FT) and the free anti-HSV-2 Fab. The samples were separated by SDS-PAGE under reducing conditions and then transferred to the nitrocellulose membrane. From left to right: marker, unbound Fab, free anti-HSV-2 Fab (C: 1 µM). Full article ">Figure 6
SPR analysis carried out on a CM7 sensor chip. (a) Scatchard Plot of the interaction between recombinant gG2 (ligand) and the NIPs (analyte). Data points were acquired starting from the 1:1 NIPs concentration and obtaining the others by successive 1:2 dilutions. (b) Sensogram of the interaction between recombinant gG2 (ligand) and the anti-HSV-2 Fab (analyte); data points were obtained at the following concentrations of the Fab: 4, 2, 1, 0.5 and 0.25 µM. Full article ">Figure 7
Cytotoxicity analysis with MTS assay on VERO cells at 24 (a) and 48 h (b). Cells were treated with four different NIPs concentrations: 50%, 25%, 12.5%, and 6.25% v/v. NPs were tested as well. The dotted red line at 70% indicates the in vitro cytotoxicity threshold. Full article ">

8 pages, 2348 KiB

Open AccessCommunication

Replication of the Venezuelan Equine Encephalitis Vaccine from a Synthetic PCR Fragment

by Christine Mathew, Colin Tucker, Irina Tretyakova and Peter Pushko

Pharmaceutics 2024, 16(9), 1217; https://doi.org/10.3390/pharmaceutics16091217 - 17 Sep 2024

Viewed by 893

Abstract

Background/Objectives: There is no approved human vaccine for Venezuelan equine encephalitis (VEE), a life-threatening disease caused by the VEE virus (VEEV). In previous studies, plasmid DNA encoding the full-length RNA genome of the VEE V4020 vaccine was used for the preparation of experimental [...] Read more.

Background/Objectives: There is no approved human vaccine for Venezuelan equine encephalitis (VEE), a life-threatening disease caused by the VEE virus (VEEV). In previous studies, plasmid DNA encoding the full-length RNA genome of the VEE V4020 vaccine was used for the preparation of experimental live virus VEE vaccines in the plasmid-transfected cell culture. Methods: Here, we used the high-fidelity polymerase chain reaction (PCR) to prepare synthetic, transcriptionally active PCR (TAP) fragments encoding the V4020 genome. Results: TAP fragment initiated the replication of the V4020 live virus vaccine in TAP fragment-transfected cells. A transfection of less than 1 ug of TAP fragment resulted in the replication of the V4020 vaccine virus in CHO cells. Conclusion: We conclude that not only plasmid DNA but also synthetic PCR-generated DNA fragments can be used for the manufacturing of live vaccines for VEEV and, potentially, other viruses. Full article

(This article belongs to the Special Issue Plasmid DNA for Gene Therapy and DNA Vaccine Applications, 2nd Edition)

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15 pages, 4287 KiB

Open AccessArticle

Targeted Delivery of STING Agonist via Albumin Nanoreactor Boosts Immunotherapeutic Efficacy against Aggressive Cancers

by Zhijun Miao, Xue Song, Anan Xu, Chang Yao, Peng Li, Yanan Li, Tao Yang and Gang Shen

Pharmaceutics 2024, 16(9), 1216; https://doi.org/10.3390/pharmaceutics16091216 - 17 Sep 2024

Viewed by 1220

Abstract

Background: Activating the cytosolic innate immune sensor, the cGAS-STING pathway, holds great promise for enhancing antitumor immunity, particularly in combination with immune checkpoint inhibitors (ICIs). However, the clinical application of STING agonists is often hindered by poor tumor accumulation, limited cellular uptake, and [...] Read more.

Background: Activating the cytosolic innate immune sensor, the cGAS-STING pathway, holds great promise for enhancing antitumor immunity, particularly in combination with immune checkpoint inhibitors (ICIs). However, the clinical application of STING agonists is often hindered by poor tumor accumulation, limited cellular uptake, and rapid clearance. To address these challenges, we developed a human serum albumin (HSA)-based nanoreactor system for the efficient delivery of the STING agonist SR-717, aiming to improve its antitumor efficacy. Methods: Using a biomineralization technique, we encapsulated SR-717 within HSA nanocages to form SH-NPs. These nanoparticles were characterized in terms of size, stability, and cellular uptake, and their ability to activate the STING pathway was assessed in both in vitro and in vivo models, including freshly isolated human renal tumor tissues. In vivo antitumor efficacy was evaluated in a murine renal tumor model, and immune responses were measured. Results: SH-NPs exhibited enhanced stability, efficient cellular uptake, and superior tumor accumulation compared to free SR-717. They robustly activated the STING pathway, as evidenced by increased phosphorylation of TBK1 and IRF3, along with elevated IFN-β production. Additionally, SH-NPs reshaped the immunosuppressive tumor microenvironment, promoting T-cell-mediated immunity and improving the therapeutic efficacy of checkpoint blockade in murine models. The validation in human renal tumor tissues further highlighted their potential for clinical translation. Importantly, SH-NPs were well tolerated with minimal systemic toxicity. Conclusions: This study underscores the potential of HSA-based nanoparticles for the targeted delivery of STING agonists, effectively enhancing antitumor immunity and improving cancer immunotherapy outcomes. SH-NPs offer a promising solution to the limitations of current STING agonists in clinical settings. Full article

(This article belongs to the Section Nanomedicine and Nanotechnology)

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Figure 1
Characterization of SH−NPs. TEM image (a) and hydrodynamic size distribution (b) of SH−NPs; (c) hydrodynamic size of SH−NPs stored at 4 °C for 7 days; (d) eta potential of SH−NPs; (e) accumulative release of SR−717 from free SR−717 and SH−NPs in pH 7.4 phosphate buffer, pH 5.0 acetate buffer, and pH 5.0 acetate buffer containing 10 μg mL−1 of CB. Full article ">Figure 2
Cellular behaviors of SH−NPs. Expression of downstream proteins of cGAS−STING signaling pathway including p−TBK1 and p−IRF3 (a) and statistics of protein relative gray value (b); (c) concentration of IFN−β secreted by cells treated with free SR−717 and SH−NPs; the expression of CD80 and CD86 on the surface of DC2.4 after administration (d) and statistics of maturation ratio (e); (f−i) expression and the fluorescence intensity of CD206 (f,g) and CD86 (h,i) on the surface of macrophages after incubation with free SR−717 and SH−NPs. Statistical differences: ** p < 0.01, *** p < 0.001 (<a href="#pharmaceutics-16-01216-f002" class="html-fig">Figure 2</a>b,c,e,g,i). Full article ">Figure 3
Tumor targeting and in vivo antitumor efficacy of SH−NPs. In vivo fluorescence imaging of renal tumor bearing mice receiving SH−NPs at different time points (a) and the fluorescence intensity of tumor region (b); (c) tumor accumulation amount of SR−717 in the renal tumor bearing mice at 12 h post−injection of SH−NPs; (d) timeline schedule of treatment of renal tumor bearing mice; (e,f) tumor growth profiles (e) and survival curve (f) of the mice bearing renal tumor treated with PBS, free SR−717, aPD−L1, SH−NPs and SH−NPs/aPD−L1. Statistical differences: *** p < 0.001 (<a href="#pharmaceutics-16-01216-f003" class="html-fig">Figure 3</a>b,c,e). Full article ">Figure 4
In vivo STING activation and immune responses. (a,b) Expression (a) and the relative protein gray value (b) of p−TBK1 and p−IRF3 in tumor tissue from the mice bearing renal tumors that were treated with PBS, free SR−717, aPD−L1, SH−NPs, and SH−NPs/aPD−L1 at 72 h post−injection; (c−f) concentration of IFN−β (c), CXCL−10 (d), IL−6 (e), TNF−α (f) in tumor tissue from the same set of mice, assessed at the same time point; (g−i) quantification of tumor−infiltrating CTLs (g,i) and NK cells (h) in tumor tissue; (j,k) quantification of T−cells (j) and Tregs (k) in tumor tissue; (i) quantification of matured dendritic cells inside tumor−draining lymph nodes; (m,n) quantification of M2−like TAMs (m) and M1−like TAMs (n) in tumor tissue; (o) quantification of MDSCs in tumor tissue. Statistical differences: * p < 0.05, ** p < 0.01, *** p < 0.001 (<a href="#pharmaceutics-16-01216-f004" class="html-fig">Figure 4</a>b−h,j−o), ns-not significant (<a href="#pharmaceutics-16-01216-f004" class="html-fig">Figure 4</a>b–e). Full article ">Figure 5
STING activation in freshly isolated human renal tumor tissues. (a−c) Concentration of IFN−β CD14b+CD68+CD86+ TAMs (b) and CD14b+CD68+CD206+ TAMs (c) in human renal tumor tissues treated with intratumoral injection of PBS, free SR−717, and SH−NPs. Statistical differences: *** p < 0.001, ns-not significant (<a href="#pharmaceutics-16-01216-f005" class="html-fig">Figure 5</a>a−c). Full article ">Scheme 1
The synthesis process of using human serum albumin (HSA) as a single−molecule nanoreactor to encapsulate the STING agonist SR−717, and the subsequent process of immune activation in tumor immunotherapy. Full article ">

21 pages, 6133 KiB

Open AccessArticle

A Patent-Pending Ointment Containing Extracts of Five Different Plants Showed Antinociceptive and Anti-Inflammatory Mechanisms in Preclinical Studies

by Juan Carlos Barragan-Galvez, Maria Leonor Gonzalez-Rivera, Juan C. Jiménez-Cruz, Araceli Hernandez-Flores, Guadalupe de la Rosa, Martha L. Lopez-Moreno, Eunice Yañez-Barrientos, Michelle Romero-Hernández, Martha Alicia Deveze-Alvarez, Pedro Navarro-Santos, Claudia Acosta-Mata, Mario Alberto Isiordia-Espinoza and Angel Josabad Alonso-Castro

Pharmaceutics 2024, 16(9), 1215; https://doi.org/10.3390/pharmaceutics16091215 - 17 Sep 2024

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Abstract

Background/Objectives: The antinociceptive and anti-inflammatory effects of a patent-pending ointment containing plant extracts from Eucalyptus globulus, Curcuma longa, Hamamelis virginiana, Echinacea purpurea, and Zingiber officinale were evaluated. Methods: Plant extracts were chemically characterized by gas chromatography–mass spectroscopy. [...] Read more.

Background/Objectives: The antinociceptive and anti-inflammatory effects of a patent-pending ointment containing plant extracts from Eucalyptus globulus, Curcuma longa, Hamamelis virginiana, Echinacea purpurea, and Zingiber officinale were evaluated. Methods: Plant extracts were chemically characterized by gas chromatography–mass spectroscopy. The antinociceptive activity of the ointment was assessed using the hot plate, tail flick, and formalin tests, whereas the anti-inflammatory activity was measured using the acute and chronic TPA-induced ear edema tests. Mechanisms of action were evaluated using inhibitors from signaling pathways related to pain response and by using histological analysis and assessing the expression and activity of pro-inflammatory mediators. Results: The ointment showed antinociceptive and anti-inflammatory effects like those observed with diclofenac gel (1.16% v/v) and ketoprofen gel (2.5% v/v). The antinociceptive actions of the ointment are mediated by the possible participation of the opiodergic system and the nitric oxide pathway. The anti-inflammatory response was characterized by a decrease in myeloperoxidase (MPO) activity and by a reduction in ear swelling and monocyte infiltration in the acute inflammation model. In the chronic model, the mechanism of action relied on a decrease in pro-inflammatory mediators such as COX-2, IL-1β, TNF-α, and MPO. An in-silico study with myristic acid, one of the compounds identified in the ointment’s plant mixture, corroborated the in vivo results. Conclusions: The ointment showed antinociceptive activities mediated by the decrease in COX-2 and NO levels, and anti-inflammatory activity due to the reduction in IL-1β and TNFα levels, a reduction in MPO activity, and a decrease in NF-κB and COX-2 expression. Full article

(This article belongs to the Section Pharmacokinetics and Pharmacodynamics)

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24 pages, 1639 KiB

Open AccessReview

Titanium Dioxide Nanomaterials: Progress in Synthesis and Application in Drug Delivery

by Fanjiao Zuo, Yameng Zhu, Tiantian Wu, Caixia Li, Yang Liu, Xiwei Wu, Jinyue Ma, Kaili Zhang, Huizi Ouyang, Xilong Qiu and Jun He

Pharmaceutics 2024, 16(9), 1214; https://doi.org/10.3390/pharmaceutics16091214 - 16 Sep 2024

Viewed by 1842

Abstract

Background: Recent developments in nanotechnology have provided efficient and promising methods for the treatment of diseases to achieve better therapeutic results and lower side effects. Titanium dioxide (TiO₂) nanomaterials are emerging inorganic nanomaterials with excellent properties such as low toxicity and [...] Read more.

Background: Recent developments in nanotechnology have provided efficient and promising methods for the treatment of diseases to achieve better therapeutic results and lower side effects. Titanium dioxide (TiO₂) nanomaterials are emerging inorganic nanomaterials with excellent properties such as low toxicity and easy functionalization. TiO₂ with special nanostructures can be used as delivery vehicles for drugs, genes and antigens for various therapeutic options. The exploration of TiO₂-based drug delivery systems shows great promise for translating nanotechnology into clinical applications; Methods: Comprehensive data on titanium dioxide were collected from reputable online databases including PubMed, GreenMedical, Web of Science, Google Scholar, China National Knowledge Infrastructure Database, and National Intellectual Property Administration; Results: In this review, we discuss the synthesis pathways and functionalization strategies of TiO₂. Recent advances of TiO₂ as a drug delivery system, including sustained and controlled drug release delivery systems were introduced. Rigorous long-term systematic toxicity assessment is an extremely critical step in application to the clinic, and toxicity is still a problem that needs to be closely monitored; Conclusions: Despite the great progress made in TiO₂-based smart systems, there is still a great potential for development. Future research may focus on developing dual-reaction delivery systems and single-reaction delivery systems like redox and enzyme reactions. Undertaking thorough in vivo investigations is necessary prior to initiating human clinical trials. The high versatility of these smart drug delivery systems will drive the development of novel nanomedicines for personalized treatment and diagnosis of many diseases with poor prognosis. Full article

(This article belongs to the Special Issue Inorganic Nanomaterials for the Development of Drug Delivery and Biomedical Applications)

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14 pages, 2871 KiB

Open AccessArticle

Synthesis and Biological Evaluation of Novel Cationic Rhenium and Technetium-99m Complexes Bearing Quinazoline Derivative for Epidermal Growth Factor Receptor Targeting

by Sotiria Triantopoulou, Ioanna Roupa, Antonio Shegani, Nektarios N. Pirmettis, Georgia I. Terzoudi, Aristeidis Chiotellis, Maria Tolia, John Damilakis, Ioannis Pirmettis and Maria Paravatou-Petsota

Pharmaceutics 2024, 16(9), 1213; https://doi.org/10.3390/pharmaceutics16091213 - 16 Sep 2024

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Abstract

Background/Objectives: Epidermal growth factor receptor (EGFR) plays a vital role in cell proliferation and survival, with its overexpression linked to various malignancies, including non-small cell lung cancer (NSCLC). Although EGFR tyrosine kinase inhibitors (TKIs) are a key therapeutic strategy, acquired resistance and relapse [...] Read more.

Background/Objectives: Epidermal growth factor receptor (EGFR) plays a vital role in cell proliferation and survival, with its overexpression linked to various malignancies, including non-small cell lung cancer (NSCLC). Although EGFR tyrosine kinase inhibitors (TKIs) are a key therapeutic strategy, acquired resistance and relapse remain challenges. This study aimed to synthesize and evaluate novel rhenium-based complexes incorporating EGFR TKIs to enhance anticancer efficacy, particularly in radiosensitization. Methods: We synthesized a rhenium tricarbonyl complex (Complex 2) and its ^99mTc analog (Complex 2’) by incorporating triphenylphosphine instead of bromine as the monodentate ligand and

{P F}_{6}^{-}

as the counter-ion, resulting in a positively charged compound that forms cationic structures. Cytotoxicity and EGFR inhibition were evaluated in A431 cells overexpressing EGFR using MTT assays, Western blotting, and flow cytometry. Radiosensitization was tested through MTT and clonogenic assays. The ^99mTc complex’s radiochemical yield, stability, and lipophilicity were also assessed. Results: Complex 2 exhibited significant cytotoxicity with an IC₅₀ of 2.6 μM and EGFR phosphorylation inhibition with an IC₅₀ of 130.6 nM. Both complex 1 and 2 induced G₀/G₁ cell cycle arrest, with Complex 2 causing apoptosis. Radiosensitization was observed at doses above 2 Gy. Complex 2’ demonstrated high stability and favorable lipophilicity (LogD_7.4 3.2), showing 12% cellular uptake after 30 min. Conclusions: Complexes 2 and 2’ show promise as dual-function anticancer agents, offering EGFR inhibition, apoptosis induction, and radiosensitization. Their potential as radiopharmaceuticals warrants further in-depth investigation in preclinical models. Full article

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

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16 pages, 656 KiB

Open AccessReview

Real-World Evidence of 3D Printing of Personalised Paediatric Medicines and Evaluating Its Potential in Children with Cancer: A Scoping Review

by Munsur Ahmed, Stephen Tomlin, Catherine Tuleu and Sara Garfield

Pharmaceutics 2024, 16(9), 1212; https://doi.org/10.3390/pharmaceutics16091212 - 14 Sep 2024

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Abstract

Personalised medicine, facilitated by advancements like 3D printing, may offer promise in oncology. This scoping review aims to explore the applicability of 3D printing for personalised pharmaceutical dosage forms in paediatric cancer care, focusing on treatment outcomes and patient experiences. Following the Joanna [...] Read more.

Personalised medicine, facilitated by advancements like 3D printing, may offer promise in oncology. This scoping review aims to explore the applicability of 3D printing for personalised pharmaceutical dosage forms in paediatric cancer care, focusing on treatment outcomes and patient experiences. Following the Joanna Briggs Institute (JBI) methodology, a comprehensive search strategy was implemented to identify the relevant literature across databases including PubMed, Embase, and Web of Science. Three independent reviewers conducted study selection and data extraction, focusing on studies involving paediatric patients under 18 years old and pharmaceutical dosage forms manufactured using 3D printing technology. From 2752 records screened, only six studies met the inclusion criteria, none of which specifically targeted paediatric cancer patients. These studies examined aspects of acceptability, including swallowability, taste, and feasibility of 3D-printed formulations for children. While the studies demonstrated the potential benefits of 3D printing in paediatric medication, particularly in personalised dosing, there is a notable lack of evidence addressing its acceptability in paediatric cancer patients. Further interdisciplinary collaborative research is needed in this area to fully assess preferences and acceptability among children with cancer and their parents or caregivers. Full article

(This article belongs to the Special Issue Advanced Pediatric Drug Formulation Strategies)

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11 pages, 4900 KiB

Open AccessCommunication

Differential Effect of Simulated Microgravity on the Cellular Uptake of Small Molecules

by Odelia Tepper-Shimshon, Nino Tetro, Roa’a Hamed, Natalia Erenburg, Emmanuelle Merquiol, Gourab Dey, Agam Haim, Tali Dee, Noa Duvdevani, Talin Kevorkian, Galia Blum, Eylon Yavin and Sara Eyal

Pharmaceutics 2024, 16(9), 1211; https://doi.org/10.3390/pharmaceutics16091211 - 14 Sep 2024

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Abstract

The space environment can affect the function of all physiological systems, including the properties of cell membranes. Our goal in this study was to explore the effect of simulated microgravity (SMG) on the cellular uptake of small molecules based on reported microgravity-induced changes [...] Read more.

The space environment can affect the function of all physiological systems, including the properties of cell membranes. Our goal in this study was to explore the effect of simulated microgravity (SMG) on the cellular uptake of small molecules based on reported microgravity-induced changes in membrane properties. SMG was applied to cultured cells using a random-positioning machine for up to three hours. We assessed the cellular accumulation of compounds representing substrates of uptake and efflux transporters, and of compounds not shown to be transported by membrane carriers. Exposure to SMG led to an increase of up to 60% (p < 0.01) in the cellular uptake of efflux transporter substrates, whereas a glucose transporter substrate showed a decrease of 20% (p < 0.05). The uptake of the cathepsin activity-based probe GB123 (MW, 1198 g/mol) was also enhanced (1.3-fold, p < 0.05). Cellular emission of molecules larger than ~3000 g/mol was reduced by up to 50% in SMG (p < 0.05). Our findings suggest that short-term exposure to SMG could differentially affect drug distribution across membranes. Longer exposure to microgravity, e.g., during spaceflight, may have distinct effects on the cellular uptake of small molecules. Full article

(This article belongs to the Special Issue Advanced Pharmaceutical Science and Technology in Israel)

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Figure 1
The uptake of efflux transporter substrates can be increased in simulated microgravity (SMG). Cells were incubated at Earth gravity or SMG (target: 0.001 g; <a href="#app3-pharmaceutics-16-01211" class="html-app">Appendix C</a>) with efflux transporter substrates in the presence or the absence of transporter inhibitors. Emission intensity was measured by flow cytometry. The left part of each panel shows a representative emission distribution curve. The right part is a quantitative analysis of the results. Shifts to the right indicate a higher uptake of the fluorescent probe by the cells. (a–c) Raw 264.7 (n = 6/group) (a), U87 (n = 4/group) (b), or MDCK-MDR1 (n = 6/group) (c) cells, incubated with 0.25 μM calcein AM with or without 1.65 μM valspodar (a P-gp inhibitor) [<a href="#B13-pharmaceutics-16-01211" class="html-bibr">13</a>]). (d) U877 cells incubated with 5 μM doxorubicin with or without 1.65 μM valspodar (n = 6/group). (e) RAW 264.7 cells incubated with 1 μM BODIPY prazosin with or without 10 μM fumitremorgin C (FTC; a BCRP inhibitor [<a href="#B13-pharmaceutics-16-01211" class="html-bibr">13</a>]; n = 6/group). (f) RAW 264.7 cells incubated with 60 μM Hoechst 33342 with or without 1.65 μM valspodar (n = 6/group, except for n = 5 in the SMG + valspodar group). Dark gray lines in the distribution curves denote non-stained cells. * p < 0.05; ** p < 0.01; 2-way ANOVA. The test assesses whether the emission is significantly affected by SMG, the inhibitor, and their interaction. The figures are labeled accordingly. Experiments were repeated thrice with similar results. The numbers of replicates per group denote biological replicates (samples cultured separately). Heights are normalized (“to mode”) to highlight shifts to the right or the left. Green, vehicle and Earth gravity; blue, vehicle and SMG; brown, transporter inhibitor and Earth gravity; purple, transporter inhibitor and SMG. Full article ">Figure 2
Simulated Moon gravity reduces the uptake of molecules larger than 3000 g/mol into RAW 264.7 cells. Cells were incubated at Earth gravity or SMG with positively charged, negatively charged, or neutral dextrans (<a href="#pharmaceutics-16-01211-t001" class="html-table">Table 1</a>) for one hour, or with NLS−PNA−FITC for three hours. Emission intensity was measured by flow cytometry. Shown are representative distribution curves of emission intensity obtained by flow cytometry (a,c) and quantitative analyses of the results (b,d). (a,b) Fluorescently labeled dextrans. (b,c) NLS−PNA−FITC. ** p < 0.01, two−way ANOVA (b) or Mann-–Whitney test (d). Experiments were repeated thrice (n = 6 biological replicates per group in each experiment), with similar results. (a,b) Green, positively-charged dextran and Earth gravity; blue, positively-charged dextran and SMG; brown, negatively-charged dextran and Earth gravity; purple, negatively-charged and SMG. Full article ">Figure A1
Placement of vials within the RPM. Full article ">Figure A2
Comparison of shelf- versus shaker placement of samples. (a) Histograms showing the distribution of cells by emission intensity within each treatment group. (b) quantitative analysis of emission intensity by group. In this experiment, calcein AM uptake into RAW 264.7 cells was assessed as described above, with additional control of “ground” samples placed on a shaker. No difference was observed between the two “ground controls” (Figure). * p < 0.05, Kruskal–Wallis test. Full article ">Figure A3
Protein loading in the cathepsin activity analysis. Full article ">Figure A4
Simulated microgravity reduces the cellular accumulation of the glucose transporter substrate 2-NBDG. RAW 264.7 cells were incubated in Earth gravity or SMG (target, 0.001 g; <a href="#app3-pharmaceutics-16-01211" class="html-app">Appendix C</a>) with 2-NBDG for one hour. Emission intensity was measured by flow cytometry. (a,b) Gating for the cells exposed to Earth (a) or SMG (b) conditions. (c). A representative distribution curve of emission intensity obtained by flow cytometry. Dark gray lines in the distribution curves denote non-stained cells. (d) Quantitative analyses of the results. n = 6/group (biological replicates); ** p < 0.01, Mann–Whitney test. Experiments were repeated twice at target 0.001 and thrice at 0.16 g with similar results. Full article ">Figure A5
Simulated Moon gravity modestly increases GB123 uptake into RAW 264.7 cells. Cells were divided into four groups, n = 6/group: two groups (1—Earth and 3—Moon) were incubated with GB123 at 0–2 h under simulated Earth or Moon conditions, washed three times, and incubated with PBS for an additional two hours (under the same respective conditions). The other groups (2—Earth and 4—Moon) were treated similarly, except that the first two hours were with the medium only (without GB123), to assess the effect of SMG on cathepsin activity. Cells were either imaged by confocal microscopy or lysed in RIPA buffer and subjected to Western blot analysis. Experiments were repeated twice, with similar results. (a,b) Confocal microscopy images of RAW 264.7 cells treated with the medium (a) or GB123 (b) for two hours. Red, GB123. Blue, DAPI. Also shown are the imaging settings. For both the Earth and the Moon images, ranges were set as 0–11,000 for the red channel, 0–14,000 for the blue channel, and 12,000–60,000 for the bright field. (c) Quantitative analysis of the GB123 signal. (d) Images of cathepsin-bound GB123 following separation on a gel. (e) Quantitative analysis of the respective GB123 emission, n = 3/group (biological replicates). The bands represent cathepsins (CTS) B, S, and L. Full article ">

14 pages, 1182 KiB

Open AccessArticle

Synthesis and Evaluation of ^99mTc(CO)₃ Complexes with Ciprofloxacin Dithiocarbamate for Infection Imaging

by Afroditi Papasavva, Nektarios N. Pirmettis, Antonio Shegani, Eleni Papadopoulou, Christos Kiritsis, Maria Georgoutsou-Spyridonos, Dimitrios C. Mastellos, Aristeidis Chiotellis, Patricia Kyprianidou, Maria Pelecanou, Minas Papadopoulos and Ioannis Pirmettis

Pharmaceutics 2024, 16(9), 1210; https://doi.org/10.3390/pharmaceutics16091210 - 14 Sep 2024

Viewed by 787

Abstract

Background: The accurate diagnosis of bacterial infections remains a critical challenge in clinical practice. Traditional imaging modalities like computed tomography (CT) and magnetic resonance imaging (MRI) often fail to distinguish bacterial infections from sterile inflammation. Nuclear medicine, such as technetium-99m (^99mTc) [...] Read more.

Background: The accurate diagnosis of bacterial infections remains a critical challenge in clinical practice. Traditional imaging modalities like computed tomography (CT) and magnetic resonance imaging (MRI) often fail to distinguish bacterial infections from sterile inflammation. Nuclear medicine, such as technetium-99m (^99mTc) radiopharmaceuticals, offers a promising alternative due to its ideal characteristics. Methods: This study explores the development of [2 + 1] mixed-ligand ^99mTc-labeled ciprofloxacin dithiocarbamate (Cip-DTC) complexes combined with various phosphine ligands, including triphenylphosphine (PPh₃), tris(4-methoxyphenyl)phosphine (TMPP), methyl(diphenyl)phosphine (MePPh₂), dimethylphenylphosphine (DMPP), and 1,3,5-triaza-7-phosphaadamantane (ADAP). The characterization of ^99mTc-complexes was conducted using rhenium analogs as structural models to ensure similar coordination. Results: Stability studies demonstrated the high integrity (97–98%) of the complexes under various conditions, including cysteine and histidine challenges. Lipophilicity studies indicated that complexes with higher logD_7.4 values (1.6–2.7) exhibited enhanced tissue penetration and prolonged circulation. Biodistribution studies in Swiss Albino mice with induced infections and aseptic inflammation revealed distinct patterns. Specifically, the complex fac-[^99mTc(CO)₃(Cip-DTC)(PPh₃)] (2′) showed high infected/normal muscle ratios (4.62 at 120 min), while the complex fac-[^99mTc(CO)₃(Cip-DTC)(TMPP)] (3′) demonstrated delayed but effective targeting (infected/normal muscle ratio of 3.32 at 120 min). Conclusions: These findings highlight the potential of ^99mTc-labeled complexes as effective radiopharmaceuticals for the differential diagnosis of bacterial infections, advancing nuclear medicine diagnostics. Future studies will focus on optimizing molecular weight, lipophilicity, and stability to further enhance the diagnostic specificity and clinical utility of these radiopharmaceuticals. Full article

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13 pages, 2078 KiB

Open AccessArticle

Assessment of Enzymatically Derived Blackcurrant Extract as Cosmetic Ingredient—Antioxidant Properties Determination and In Vitro Diffusion Study

by Anja Petrov Ivanković, Marija Ćorović, Ana Milivojević, Stevan Blagojević, Aleksandra Radulović, Rada Pjanović and Dejan Bezbradica

Pharmaceutics 2024, 16(9), 1209; https://doi.org/10.3390/pharmaceutics16091209 - 14 Sep 2024

Cited by 1 | Viewed by 1003

Abstract

Blackcurrant is an anthocyanin-rich berry with proven antioxidant and photoprotective activity and emerging prebiotic potential, widely applied in cosmetic products. Hereby, highly efficient enzyme-assisted extraction of blackcurrant polyphenols was performed, giving extract with very high antioxidant activity. Obtained extract was characterized in terms [...] Read more.

Blackcurrant is an anthocyanin-rich berry with proven antioxidant and photoprotective activity and emerging prebiotic potential, widely applied in cosmetic products. Hereby, highly efficient enzyme-assisted extraction of blackcurrant polyphenols was performed, giving extract with very high antioxidant activity. Obtained extract was characterized in terms of anthocyanin composition, incorporated into three different cosmetic formulations and subjected to Franz cell diffusion study. Experimental values obtained using cellulose acetate membrane for all four dominant anthocyanins (delphinidin 3-glucoside, delphinidin 3-rutinoside, cyanidin 3-glucoside and cyanidin 3-rutinoside) were successfully fitted with the Korsmeyer–Peppas diffusion model. Calculated effective diffusion coefficients were higher for hydrogel compared to oil-in-water cream gel and oil-in-water emulsion, whereas the highest value was determined for cyanidin 3-rutinoside. On the other hand, after a 72 h long experiment with transdermal skin diffusion model (Strat-M^® membrane), no anthocyanins were detected in the receptor fluid, and only 0.5% of the initial quantity from the donor compartment was extracted from the membrane itself after experiment with hydrogel. Present study revealed that hydrogel is a suitable carrier system for the topical delivery of blackcurrant anthocyanins, while dermal and transdermal delivery of these molecules is very limited, which implies its applicability for treatments targeting skin surface (i.e., prebiotic, photoprotective). Full article

(This article belongs to the Special Issue Advances in Natural Products for Cutaneous Application)

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