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Pharmaceutics
Volume 16
Issue 3
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Pharmaceutics, Volume 16, Issue 3 (March 2024) – 140 articles

Cover Story (view full-size image): The opportunistic bacteria growing in biofilms play a decisive role in the pathogenesis of chronic infectious diseases. Antimicrobial adjuvants have emerged as a promising strategy to combat antimicrobial resistance (AMR) and restore the efficacy of existing antibiotics. The co-delivery of antibiotics and potential antimicrobial adjuvants through encapsulation in lipid nanocarriers (LNCs) can improve the effects of antibiotics and potentially reduce bacterial resistance owing to the superior properties of LNCs compared to traditional formulations. This review discusses how LNC-enabled co-delivery of antibiotics and adjuvants can advance current clinical antimicrobial treatments, leading to innovative products, enabling the reuse of antibiotics, and providing opportunities to save millions of lives from bacterial infections. View this paper
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21 pages, 1750 KiB  
Article
Optimization of the Production Process of Clinical-Grade Human Salivary Gland Organoid-Derived Cell Therapy for the Treatment of Radiation-Induced Xerostomia in Head and Neck Cancer
by Jacoba van Zanten, Annelies Jorritsma-Smit, Hans Westra, Mirjam Baanstra, Anne de Bruin-Jellema, Derk Allersma, Bahez Gareb and Rob P. Coppes
Pharmaceutics 2024, 16(3), 435; https://doi.org/10.3390/pharmaceutics16030435 - 21 Mar 2024
Cited by 6 | Viewed by 2107
Abstract
Head and neck cancer is a common cancer worldwide. Radiotherapy has an essential role in the treatment of head and neck cancers. After irradiation, early effects of reduced saliva flow and hampered water secretion are seen, along with cell loss and a decline [...] Read more.
Head and neck cancer is a common cancer worldwide. Radiotherapy has an essential role in the treatment of head and neck cancers. After irradiation, early effects of reduced saliva flow and hampered water secretion are seen, along with cell loss and a decline in amylase production. Currently, there is no curative treatment for radiation-induced hyposalivation/xerostomia. This study aimed to develop and optimize a validated manufacturing process for salivary gland organoid cells containing stem/progenitor cells using salivary gland patient biopsies as a starting material. The manufacturing process should comply with GMP requirements to ensure clinical applicability. A laboratory-scale process was further developed into a good manufacturing practice (GMP) process. Clinical-grade batches complying with set acceptance and stability criteria were manufactured. The results showed that the manufactured salivary gland-derived cells were able to self-renew, differentiate, and show functionality. This study describes the optimization of an innovative and promising novel cell-based therapy. Full article
(This article belongs to the Special Issue Pharmacy Compounding of Personalized Preparation for Specific Patients: Challenges and Advantages)
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<p>(<b>A</b>): The number of cells after P0, P1, and P2 in the technology transfer batches TTR1-6. (<b>B</b>): The sphere-forming potency of the technology transfer batches TTR 1–TTR 6.</p> Full article ">Figure 2
<p>(<b>A</b>): The number of cells after P0 and P1 in the technology transfer batches TTR 7–TTR 9. (<b>B</b>): The sphere-forming potency of the technology transfer batches TTR 7–TTR 9.</p> Full article ">Figure 3
<p>(<b>A</b>): The number of cells after P0 and P1 in the technology transfer batches TTR 10–TTR 11. (<b>B</b>): The sphere-forming potency of the technology transfer batches TTR 10–TTR 11.</p> Full article ">Figure 4
<p>Representative images of the differentiated cells forming duct-like structures. Single cell-derived salispheres derived from P1 cultures of technology transfer batches TTR 7 (<b>A</b>,<b>B</b>) or TTR 8 (<b>C</b>,<b>D</b>) were differentiated in the gel for up to three weeks. Phase contrast microscopy pictures were taken at the end of the culture period. The images show the formation of organoids with salivary gland structures, including branching and the formation of lobular structures (scale bars in images (<b>B</b> ) and (<b>D</b>) 500 µm).</p> Full article ">Figure 5
<p>Representative images of the staining of the duct-like structures. (<b>A</b>): Alcian blue staining. (<b>B</b>): PAS staining. (<b>C</b>): Amylase staining. (<b>D</b>): CK19 staining.</p> Full article ">
17 pages, 5859 KiB  
Article
Maleimide–Thiol Linkages Alter the Biodistribution of SN38 Therapeutic Microbubbles Compared to Biotin–Avidin While Preserving Parity in Tumoral Drug Delivery
by Nicola Ingram, Radwa H. Abou-Saleh, Amanda D. Race, Paul M. Loadman, Richard J. Bushby, Stephen D. Evans and P. Louise Coletta
Pharmaceutics 2024, 16(3), 434; https://doi.org/10.3390/pharmaceutics16030434 - 21 Mar 2024
Viewed by 1985
Abstract
Therapeutic microbubbles (thMBs) contain drug-filled liposomes linked to microbubbles and targeted to vascular proteins. Upon the application of a destructive ultrasound trigger, drug uptake to tumour is improved. However, the structure of thMBs currently uses powerful non-covalent bonding of biotin with avidin-based proteins [...] Read more.
Therapeutic microbubbles (thMBs) contain drug-filled liposomes linked to microbubbles and targeted to vascular proteins. Upon the application of a destructive ultrasound trigger, drug uptake to tumour is improved. However, the structure of thMBs currently uses powerful non-covalent bonding of biotin with avidin-based proteins to link both the liposome to the microbubble (MB) and to bind the targeting antibody to the liposome–MB complex. This linkage is not currently FDA-approved, and therefore, an alternative, maleimide–thiol linkage, that is currently used in antibody–drug conjugates was examined. In a systematic manner, vascular endothelial growth factor receptor 2 (VEGFR2)-targeted MBs and thMBs using both types of linkages were examined for their ability to specifically bind to VEGFR2 in vitro and for their ultrasound imaging properties in vivo. Both showed equivalence in the production of the thMB structure, in vitro specificity of binding and safety profiles. In vivo imaging showed subtle differences for thMBs where biotin thMBs had a faster wash-in rate than thiol thMBs, but thiol thMBs were longer-lived. The drug delivery to tumours was also equivalent, but interestingly, thiol thMBs altered the biodistribution of delivery away from the lungs and towards the liver compared to biotin thMBs, which is an improvement in biosafety. Full article
(This article belongs to the Special Issue Lipid-Based Nanoparticles for Drug Delivery in Cancer)
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Graphical abstract

Graphical abstract
Full article ">Figure 1
<p>Thiol and maleimide Functionalisation. Thiol functionality of lipids was provided either (1) by incorporating PDTE into the phospholipid layer or (2) by incorporating a PDP lipid which was reduced with TCEP. (3) Thiol functionality of proteins was achieved by reacting with Traut’s reagent. (4) Linkage of the these was achieved by a reversible reaction of the thiol functionalised phospholipids or proteins with lipids that were functionalised with maleimide, which strongly favours the generation of the thiol-maleimide linkage.</p> Full article ">Figure 2
<p>Liposomes can be bound to MBs using maleimide–thiol linkages to the same degree as biotin–avidin linkages. (<b>a</b>,<b>b</b>) Linkages using either maleimide liposomes with PDP-containing MBs or PDP liposomes with maleimide-containing MBs are feasible. The fluorescence detected around the MBs shows loading of the liposomes on the MB surface. Scale bar denotes 10 μm. (<b>c</b>) Biotin and maleimide MBs were generated with green fluorescent lipid in their shell and the SN38-encapsulating liposome (biotin or PtD) was generated with red fluorescent lipid in their shell. Example flow cytometry plots gated on the MB population show the fluorescence from biotin liposome-loaded MBs (left) or thiol liposome-loaded MBs (right). The pie charts show the percentage of all MBs with liposomes bound to them (green +, red +) as determined by flow cytometry (<b>d</b>).</p> Full article ">Figure 3
<p>Antibodies can be thiolated using Traut’s reagent and retain their antigen recognition properties. The anti-mouse VEGFR2 antibody, Avas12a1 was thiolated using Traut’s reagent. Ellman’s assay quantified the amount of free sulfhydryl with decreasing concentrations of input antibody (<b>a</b>) and over incubation time with the Traut’s reagent using 3 mg/mL antibody (<span class="html-italic">n</span> = 3, mean and standard deviation are shown) (<b>b</b>). Mouse endothelial SVR cells were incubated with thiolated (<b>c</b>) or non-thiolated (<b>d</b>) mouse anti-VEGFR2 antibody to examine any difference in staining patterns that would indicate disruption of VEGFR2 recognition.</p> Full article ">Figure 4
<p>Schematic of the generation of thMBs using biotin–avidin and maleimide–thiol linkages. (<b>a</b>) Drug-loaded liposomes incorporating biotinylated lipids are generated by thin film hydration and extrusion. Liposomes are rehydrated after freeze drying and incubated with NeutrAvidin. Liposomes are then mixed with MB shell lipids in buffer. Microfluidics are used to generate liposome-loaded gas-filled MBs. These are incubated with commercially available biotinylated antibodies to generate thMBs. (<b>b</b>) Drug-loaded liposomes incorporating PtD lipids are generated by thin film hydration and extrusion and a small amount of TCEP is added before freezing. Liposomes are rehydrated after freeze drying and mixed with MB shell lipids in buffer. Microfluidics are used to generate liposome-loaded gas-filled MBs. Separately, the antibody is thiolated using Traut’s reagent. These antibodies are incubated with the liposome-loaded MBs to generate thMBs.</p> Full article ">Figure 5
<p>Thiolated antibodies on MBs or thMBs show good targeting specificity in vitro. (<b>a</b>) VEGFR2- or isotype-targeted MBs were bound to mouse SVR endothelial cells under fluid flow. Biotin–avidin linkages and maleimide–thiol linkages for both VEGFR2-targeted MBs and thMBs were compared to isotype, unpaired <span class="html-italic">t</span>-test, two-tailed). <span class="html-italic">n</span> = 3 experimental replicates; mean and standard deviation are shown. The frequency of single or multiple targeted MBs bound per cell was plotted for isotype or targeted antibodies with biotin linkages (<b>b</b>) or thiol linkages (<b>c</b>).</p> Full article ">Figure 6
<p>Significant haemolysis was not observed with either biotinylated or thiolated MB architectures and showed no significant differences in cytotoxicity in vitro. (<b>a</b>) Liposome-loaded MBs were incubated with red blood cells from 5 mice in triplicate. The amount of haemolysis observed was quantitated compared to a positive control (addition of Triton X-100-treated red blood cells). <span class="html-italic">n</span> = 3, mean and standard deviation are shown. Cells were incubated with SN38 liposome-loaded MBs and the reduction in cell viability was measured in SW480 (<b>b</b>) and SW620 (<b>c</b>) cells. The graph shows the mean and standard deviation in cell viability compared to vehicle, <span class="html-italic">n</span> = 3.</p> Full article ">Figure 7
<p>In vivo imaging showing subtle differences in imaging parameters between biotin–avidin and maleimide–thiol MBs and thMBs. (<b>a</b>) Parameters of the time intensity curve, generated by MB injection, were examined for differences in peak enhancement (P.E.) (<b>b</b>), time to peak (<b>c</b>) and the wash-in rate (<b>d</b>) between biotinylated or thiolated targeted MBs or thMBs. (<b>e</b>) The lifetime of the MB architectures was also examined in terms of the area under the curve (AUC) (<b>f</b>), the length of time that the contrast-enhanced signal intensity was detected at half the maximum signal or above (FWHM—(<b>g</b>)), and the wash-out rate (<b>h</b>). Individual data points represent a single mouse, the median is denoted, * <span class="html-italic">p</span> &lt; 0.05, Mann–Whitney, two-tailed.</p> Full article ">Figure 8
<p>In vivo imaging differences in biotinylated and thiolated MB architectures are not due to differences in physiology between animals. (<b>a</b>) Doppler velocities (mm/s) were measured per animal using pulsed wave Doppler signal in the aorta alongside velocity time integral (cm/stroke, <b>b</b>), depth of the aorta (mm, <b>c</b>), mean heart rate at the beginning and end of the imaging process (beats per minute, bpm, <b>d</b>) and the same for the respiration rate (bpm, <b>e</b>). The results are shown for individual mice per MB type. ANOVA followed by Sidak’s multiple comparisons showed no statistically significant differences between the MB types for any of the physiological parameters measured.</p> Full article ">Figure 9
<p>In vivo drug delivery of the two MB architectures showing differences in biodistribution. LC-MS/MS quantitation of tumour and organs from mice injected with thMBs that contained either biotin–avidin or maleimide–thiol linkages. The concentration of the encapsulated drug, SN38 (<b>a</b>), and its glucuronidated metabolite, SN38G (<b>b</b>), was determined in each tissue. <span class="html-italic">n</span> = 5 mice per group with the median denoted. Two-way ANOVA followed by Sidak’s multiple comparisons is shown **** <span class="html-italic">p</span> &lt; 0.0001. LOQ means the sample was below the limit of quantitation (10 pg/mL for both SN38 and SN38G).</p> Full article ">
20 pages, 12958 KiB  
Article
Triboelectric Charging Properties of the Functional Groups of Common Pharmaceutical Materials Using Density Functional Theory Calculations
by James R. Middleton, Mojtaba Ghadiri and Andrew J. Scott
Pharmaceutics 2024, 16(3), 433; https://doi.org/10.3390/pharmaceutics16030433 - 21 Mar 2024
Viewed by 1567
Abstract
Triboelectrification is a ubiquitous and poorly understood phenomenon in powder processing, particularly for pharmaceutical powders. Charged particles can adhere to vessel walls, causing sheeting; they can also cause agglomeration, threatening the stability of powder formulations, and in extreme cases electrostatic discharges, which present [...] Read more.
Triboelectrification is a ubiquitous and poorly understood phenomenon in powder processing, particularly for pharmaceutical powders. Charged particles can adhere to vessel walls, causing sheeting; they can also cause agglomeration, threatening the stability of powder formulations, and in extreme cases electrostatic discharges, which present a serious fire and explosion hazard. Triboelectrification is highly sensitive to environmental and material conditions, which makes it very difficult to compare experimental results from different publications. In this work, density functional theory (DFT) is used to investigate the charge transfer characteristics of several functional groups of paracetamol in order to better understand the mechanisms of charging at the nanoscale and the influence of the environmental and material properties on charge transfer. This is achieved by studying the structure and electronic properties at the molecule–substrate interface. Using this molecule–substrate approach, the charging contributions of individual functional groups are explored by examining the Hirschfeld charges, the charge density difference between the molecule and substrate, the density of states, and the location of the frontier orbitals (HOMO and LUMO) of a paracetamol molecule. Charge density difference calculations indicate a significant transfer of charge from the molecule to the surface. Observable regions of electron density enrichment and depletion are evident around the electron-donating and -withdrawing groups, respectively. The density of states for the paracetamol molecule evolves as it approaches the surface, and the band gap disappears upon contact with the substrate. Hirshfeld charge analysis reveals asymmetry in the charge redistribution around the molecule, highlighting the varying charging tendencies of different atoms. Full article
(This article belongs to the Special Issue 15th Anniversary of Pharmaceutics—Advances in Process and Formulation Modeling)
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Figure 1

Figure 1
<p>Bulk unit cell of aluminium, copper, nickel, and paracetamol.</p> Full article ">Figure 2
<p>Sensitivity analysis of Hirshfeld charge versus number of atomic surface layers, carried out on an aluminium (100) surface, using a 600 eV kinetic energy cutoff, and 2 × 2 × 1 Monkhorst–Pack grid.</p> Full article ">Figure 3
<p>Electrostatic potential of aluminium (111) surface with a single paracetamol placed on its surface at its structurally optimised position. Electrostatic potential of the isolated surface and isolated molecule also shown.</p> Full article ">Figure 4
<p>Charge density difference iso-surface of paracetamol on nickel (<b>left</b>), copper (<b>middle</b>), and aluminium (<b>right</b>) at the geometry-optimised position from the surface, <math display="inline"><semantics> <mrow> <msub> <mrow> <mi>d</mi> </mrow> <mrow> <mn>0</mn> </mrow> </msub> </mrow> </semantics></math>. Top view. Iso value = 7 × 10<sup>−3</sup> electron/<math display="inline"><semantics> <mrow> <mi mathvariant="normal">Å</mi> </mrow> </semantics></math><sup>3</sup>.</p> Full article ">Figure 5
<p>Charge density difference iso-surface of paracetamol on aluminium (111) surface at several distances. Side view. The geometry-optimised position of paracetamol is indicated by <math display="inline"><semantics> <mrow> <msub> <mrow> <mi>d</mi> </mrow> <mrow> <mn>0</mn> </mrow> </msub> </mrow> </semantics></math>. Iso value = 7 × 10<sup>−3</sup> electron/<math display="inline"><semantics> <mrow> <mi mathvariant="normal">Å</mi> </mrow> </semantics></math><sup>3</sup>.</p> Full article ">Figure 6
<p>Planar-averaged charge density difference of the aluminium–paracetamol system at several distances.</p> Full article ">Figure 7
<p>Planar-averaged charge density difference of the copper–paracetamol system at several distances.</p> Full article ">Figure 8
<p>Planar-averaged charge density difference of the nickel–paracetamol system at several distances.</p> Full article ">Figure 9
<p>DOS of a paracetamol 5 Å away from its optimised position on an aluminium (111) surface (<b>left</b>). Iso-surface of HOMO (red) and LUMO (blue) orbitals obtained from DOS calculation on molecule (<b>right</b>).</p> Full article ">Figure 10
<p>Evolution of the DOS of paracetamol as the molecule is withdrawn from an aluminium (111) surface.</p> Full article ">Figure 11
<p>Comparison of the density of states (DOS) for paracetamol at the optimised distance from an aluminium (111) surface and at a distance of 5 Å away from the optimised position on the surface.</p> Full article ">Figure 12
<p>Comparison of the density of states (DOS) for paracetamol at the optimised distance from a copper (111) surface and at a distance of 5 Å away from the optimised position on the surface.</p> Full article ">Figure 13
<p>Comparison of the density of states (DOS) for paracetamol at the optimised distance from a nickel (111) surface and at a distance of 5 Å away from the optimised position on the surface.</p> Full article ">Figure 14
<p>Hirshfeld charges mapped onto their respective atoms. Aluminium (<b>top left</b>). Copper (<b>top right</b>). Nickel (<b>bottom</b>). Side view.</p> Full article ">Figure 15
<p>Surface Hirshfeld charges mapped on to their respective surface atoms. Aluminium (<b>left</b>). Copper (<b>middle</b>). Nickel (<b>right</b>). Bottom view.</p> Full article ">Figure 16
<p>Hirshfeld charges of paracetamol molecule. Isolated (<b>top left</b>), on aluminium surface (<b>top right</b>), on copper surface (<b>bottom left</b>), on nickel surface (<b>bottom right</b>).</p> Full article ">Figure 17
<p>Hirshfeld charges on each atom of an isolated paracetamol molecule.</p> Full article ">Figure 18
<p>Hirshfeld charges on each atom of a paracetamol molecule placed on a (111) metal surface.</p> Full article ">Figure 19
<p>Difference between Hirshfeld charge at the surface and the Hirshfeld charge 5 Å from the surface.</p> Full article ">
18 pages, 1826 KiB  
Review
Polymer-Drug Anti-Thrombogenic and Hemocompatible Coatings as Surface Modifications
by Barbara Zawidlak-Węgrzyńska, Joanna Rydz, Marta Musioł and Aneta Radziwon-Balicka
Pharmaceutics 2024, 16(3), 432; https://doi.org/10.3390/pharmaceutics16030432 - 21 Mar 2024
Cited by 1 | Viewed by 2585
Abstract
Since the 1960s, efforts have been made to develop new technologies to eliminate the risk of thrombosis in medical devices that come into contact with blood. Preventing thrombosis resulting from the contact of a medical device, such as an implant, with blood is [...] Read more.
Since the 1960s, efforts have been made to develop new technologies to eliminate the risk of thrombosis in medical devices that come into contact with blood. Preventing thrombosis resulting from the contact of a medical device, such as an implant, with blood is a challenge due to the high mortality rate of patients and the high cost of medical care. To this end, various types of biomaterials coated with polymer-drug layers are being designed to reduce their thrombogenicity and improve their hemocompatibility. This review presents the latest developments in the use of polymer-drug systems to produce anti-thrombogenic surfaces in medical devices in contact with blood, such as stents, catheters, blood pumps, heart valves, artificial lungs, blood vessels, blood oxygenators, and various types of tubing (such as for hemodialysis) as well as microfluidic devices. This paper presents research directions and potential clinical applications, emphasizing the importance of continued progress and innovation in the field. Full article
(This article belongs to the Special Issue Advances in Polymeric Drug Delivery Systems)
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<p>A drug-eluting stent (DES) scheme. Originally published in ref. [<a href="#B52-pharmaceutics-16-00432" class="html-bibr">52</a>] under CC BY 4.0 license.</p> Full article ">Figure 2
<p>Rotary blood pump: (<b>a</b>) schematic of the blood pump design; (<b>b</b>) housing model. Originally published in ref. [<a href="#B91-pharmaceutics-16-00432" class="html-bibr">91</a>] under CC BY 4.0 license.</p> Full article ">Figure 3
<p>Diagram of the extracorporeal membrane oxygenation model: (<b>a</b>) diagram of the veno-arterial system; (<b>b</b>) diagram of the veno-venous system. Originally published in ref. [<a href="#B103-pharmaceutics-16-00432" class="html-bibr">103</a>] under CC BY 4.0 license.</p> Full article ">Figure 4
<p>Diagram of the dialysis process. Originally published in ref. [<a href="#B112-pharmaceutics-16-00432" class="html-bibr">112</a>] under CC BY 4.0 license.</p> Full article ">
38 pages, 7322 KiB  
Systematic Review
Targeting the Gut: A Systematic Review of Specific Drug Nanocarriers
by Patrizia Garbati, Cristiana Picco, Raffaella Magrassi, Paolo Signorello, Ludovica Cacopardo, Mauro Dalla Serra, Maria Grazia Faticato, Maria De Luca, Francesco Balestra, Maria Principia Scavo and Federica Viti
Pharmaceutics 2024, 16(3), 431; https://doi.org/10.3390/pharmaceutics16030431 - 21 Mar 2024
Cited by 6 | Viewed by 3999
Abstract
The intestine is essential for the modulation of nutrient absorption and the removal of waste. Gut pathologies, such as cancer, inflammatory bowel diseases (IBD), irritable bowel syndrome (IBS), and celiac disease, which extensively impact gut functions, are thus critical for human health. Targeted [...] Read more.
The intestine is essential for the modulation of nutrient absorption and the removal of waste. Gut pathologies, such as cancer, inflammatory bowel diseases (IBD), irritable bowel syndrome (IBS), and celiac disease, which extensively impact gut functions, are thus critical for human health. Targeted drug delivery is essential to tackle these diseases, improve therapy efficacy, and minimize side effects. Recent strategies have taken advantage of both active and passive nanocarriers, which are designed to protect the drug until it reaches the correct delivery site and to modulate drug release via the use of different physical–chemical strategies. In this systematic review, we present a literature overview of the different nanocarriers used for drug delivery in a set of chronic intestinal pathologies, highlighting the rationale behind the controlled release of intestinal therapies. The overall aim is to provide the reader with useful information on the current approaches for gut targeting in novel therapeutic strategies. Full article
(This article belongs to the Special Issue Nanotechnology-Based Pharmaceutical Treatments)
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<p>Schematic overview of nanocarrier size.</p> Full article ">Figure 2
<p>Simplified schema of composition of organic and inorganic drug nanocarriers. In the upper panel (organic nanocarrier), liposomes appear modified with molecules, such as PEG + functionalizing layer, antibodies with cleavable sites, carbohydrates, and hydrophobic/hydrophilic drugs. Other organic carriers are reported (EVs and lipid nanoparticles). Inorganic nanocarriers (lower panel) can be modified with antibodies and/or drugs. In particular, drugs can be contained inside (such as in microspheres) or linked outside (such as in SiNPs) the carrier. Moreover, modifications can adhere to the surface of the nanoparticles by means of surfactants or they could be packaged in the pores (such as in the case of multistage vectors).</p> Full article ">Figure 3
<p>Illustration of pH variants in the intestinal tract, with specific focus on the structure of the large and small intestines.</p> Full article ">Figure 4
<p>Overall strategy implemented in this review.</p> Full article ">Figure 5
<p>Flowchart of the inclusion/exclusion process leading to the final number of analyzed papers. Integral systems include microspheres and SEDDS/SNEDDS/SMEDDS.</p> Full article ">Figure 6
<p>Summary of intestine delivery strategies.</p> Full article ">
24 pages, 3582 KiB  
Review
Synthesis and Properties of Injectable Hydrogel for Tissue Filling
by Chunyu Xie, Ga Liu, Lingshuang Wang, Qiang Yang, Fuying Liao, Xiao Yang, Bo Xiao and Lian Duan
Pharmaceutics 2024, 16(3), 430; https://doi.org/10.3390/pharmaceutics16030430 - 21 Mar 2024
Cited by 11 | Viewed by 2871
Abstract
Hydrogels with injectability have emerged as the focal point in tissue filling, owing to their unique properties, such as minimal adverse effects, faster recovery, good results, and negligible disruption to daily activities. These hydrogels could attain their injectability through chemical covalent crosslinking, physical [...] Read more.
Hydrogels with injectability have emerged as the focal point in tissue filling, owing to their unique properties, such as minimal adverse effects, faster recovery, good results, and negligible disruption to daily activities. These hydrogels could attain their injectability through chemical covalent crosslinking, physical crosslinking, or biological crosslinking. These reactions allow for the formation of reversible bonds or delayed gelatinization, ensuring a minimally invasive approach for tissue filling. Injectable hydrogels facilitate tissue augmentation and tissue regeneration by offering slow degradation, mechanical support, and the modulation of biological functions in host cells. This review summarizes the recent advancements in synthetic strategies for injectable hydrogels and introduces their application in tissue filling. Ultimately, we discuss the prospects and prevailing challenges in developing optimal injectable hydrogels for tissue augmentation, aiming to chart a course for future investigations. Full article
(This article belongs to the Special Issue Design of Injectable and Self-Healing Hydrogels for Tissue Regeneration and Drug Delivery)
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<p>Schematic of chemical covalent crosslinked injectable hydrogel via (<b>A</b>) reversable bonding based on Michael addition method [<a href="#B30-pharmaceutics-16-00430" class="html-bibr">30</a>], (<b>B</b>) reversable bonding based on borate imine and borate bonds [<a href="#B45-pharmaceutics-16-00430" class="html-bibr">45</a>], (<b>C</b>) delayed gelatinization based on radical polymerization [<a href="#B48-pharmaceutics-16-00430" class="html-bibr">48</a>], (<b>D</b>) delayed gelatinization based on thiolated HA/CMC [<a href="#B52-pharmaceutics-16-00430" class="html-bibr">52</a>], (<b>E</b>) delayed gelatinization based on Schiff base reaction [<a href="#B54-pharmaceutics-16-00430" class="html-bibr">54</a>] and (<b>F</b>) delayed gelatinization based on Diels–Alder reaction [<a href="#B56-pharmaceutics-16-00430" class="html-bibr">56</a>]. Reproduced with permission.</p> Full article ">Figure 2
<p>Schematic of physical crosslinked injectable hydrogel via (<b>A</b>) reversable bonds based on hydrogen bonding [<a href="#B63-pharmaceutics-16-00430" class="html-bibr">63</a>], (<b>B</b>) reversable bonds based on hydrophobic interactions [<a href="#B70-pharmaceutics-16-00430" class="html-bibr">70</a>], (<b>C</b>) reversable bonds based on host−guest interaction [<a href="#B75-pharmaceutics-16-00430" class="html-bibr">75</a>] and (<b>D</b>) reversable bonds based on multiple physical interactions (including hydrogen bonding and ions coordination) [<a href="#B85-pharmaceutics-16-00430" class="html-bibr">85</a>]. Reproduced with permission.</p> Full article ">Figure 3
<p>Schematic of (<b>A</b>) biological crosslinked injectable hydrogel via delayed gelatinization based on the catalyzation by horseradish peroxidase [<a href="#B89-pharmaceutics-16-00430" class="html-bibr">89</a>] and (<b>B</b>) injectable microgel crosslinked by transglutaminase [<a href="#B91-pharmaceutics-16-00430" class="html-bibr">91</a>]. Reproduced with permission.</p> Full article ">Figure 4
<p>(<b>A</b>) The injection force of SF-HA and CaHA-CMC measured with a crosshead speed of 13 mm/min. (<b>B</b>) Hematoxylin and eosin staining (H&amp;E) of tissue round the SF-HA and CaHA-CMC injected site (scale bar = 125 µm). Cellular infiltration, predominantly comprising macrophages and giant cells responsible for the enzymatic degradation of silk protein, is observed in proximity to silk particles (yellow arrows). Cross-sections of silk-HA exhibit vascularity (green arrows), within the tissue ingrowth. Areas of HA (blue arrows) demonstrate cell occlusion and undergo collapse during histological processing. Similarly, CaHA-CMC facilitates the infiltration of macrophages and giant cells in areas adjacent to CaHA particles (black arrows). Sections from both 9- and 12-month intervals were subjected to decalcification prior to staining, leading to the formation of "ghost" regions where CaHA particles were once located. Analogous to HA, CMC (orange arrows) exhibits cell occlusive properties. Reproduced with permission [<a href="#B128-pharmaceutics-16-00430" class="html-bibr">128</a>].</p> Full article ">Figure 5
<p>(<b>A</b>) H&amp;E staining images and (<b>B</b>) immunostaining images of skin tissues injected with the PF127, levan/PF127, HA/CMC/PF127 and levan CMC/PF-127 (scale bar = 200 µm). (<b>C</b>) Schematic image for the construction of the hairless mice model with the skin wrinkling and the schedule for injection. (<b>D</b>) Skin surface images after injection with different hydrogel. Reproduced with permission [<a href="#B149-pharmaceutics-16-00430" class="html-bibr">149</a>].</p> Full article ">
12 pages, 3738 KiB  
Article
Mannose-Decorated Solid-Lipid Nanoparticles for Alveolar Macrophage Targeted Delivery of Rifampicin
by Hriday Bera, Caizhu Zhao, Xidong Tian, Dongmei Cun and Mingshi Yang
Pharmaceutics 2024, 16(3), 429; https://doi.org/10.3390/pharmaceutics16030429 - 20 Mar 2024
Cited by 2 | Viewed by 2440
Abstract
Alveolar macrophages play a vital role in a variety of lung diseases, including tuberculosis. Thus, alveolar macrophage targeted anti-tubercular drug delivery through nanocarriers could improve its therapeutic response against tuberculosis. The current study aimed at exploring the efficacy of glyceryl monostearate (GMS)-based solid-lipid [...] Read more.
Alveolar macrophages play a vital role in a variety of lung diseases, including tuberculosis. Thus, alveolar macrophage targeted anti-tubercular drug delivery through nanocarriers could improve its therapeutic response against tuberculosis. The current study aimed at exploring the efficacy of glyceryl monostearate (GMS)-based solid-lipid nanoparticles (SLNs) and their mannose functionalized forms on the alveolar macrophage targeting ability of an anti-tubercular model drug, rifampicin (Rif). Rif-loaded SLNs were accomplished by the solvent diffusion method. These carriers with unimodal particle size distribution (~170 nm) were further surface-modified with mannose via Schiff-base reaction, leading to slight enhancement of particle diameter and a decline of drug loading capacity. The encapsulated Rif, which was molecularly dispersed within the matrices as indicated by their XRD patterns, was eluted in a sustained manner with an initial burst release effect. The uptake efficiency of mannose-modified SLNs was remarkably higher than that of corresponding native forms on murine macrophage Raw 264.7 cells and human lung adenocarcinoma A549 cells. Eventually, the mannose-modified SLNs showed a greater cytotoxicity on Raw 264.7 and A549 cells relative to their unmodified forms. Overall, our study demonstrated that mannose modification of SLNs had an influence on their uptake by alveolar macrophages, which could provide guidance for the future development of alveolar macrophage targeted nanoformulations. Full article
(This article belongs to the Section Nanomedicine and Nanotechnology)
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<p>Schematic illustration of passive and active targeting of SLNs to macrophages (<b>A</b>). Preparation schemes of the Rif-SLNs (<b>B</b>) and Rif-Man-SLNs (<b>C</b>).</p> Full article ">Figure 2
<p>Physical appearances of blank SLNs (<b>left</b>) and Rif-SLNs (<b>right</b>) (<b>A</b>) and blank Man-SLNs (<b>left</b>) and Rif-Man-SLNs (<b>right</b>) (<b>B</b>). TEM images of Rif-SLNs (<b>C</b>) and Rif-Man-SLNs (<b>D</b>).</p> Full article ">Figure 3
<p>FT-IR overlay spectra of mannose, SA, GMS, physical mixture, SLNs, Man–SLNs (<b>A</b>). XRD patterns of Rif, GMS, physical mixture, Rif–SLNs (<b>B</b>) and XRD patterns of Rif, GMS, SA, mannose, physical mixture, Rif–Man–SLNs (<b>C</b>).</p> Full article ">Figure 4
<p>The stability of pure Rif in PBS media containing different amounts of VC (<b>A</b>). The in vitro release profiles of Rif from Rif-SLNs and Rif-Man-SLNs in PBS containing 200 μg/mL of VC (<b>B</b>) (<span class="html-italic">n</span> = 3, Mean ± SD).</p> Full article ">Figure 5
<p>Cellular internalization of C6-SLNs, C6-SA-SLNs and C6-Man-SLNs on Raw 264.7 cells (<b>A</b>) and A549 cells (<b>B</b>) analyzed using CLSM. Cellular uptake efficiency of SLNs recorded by flow cytometry (<b>C</b>,<b>D</b>,<b>F</b>,<b>G</b>) and quantitative evaluation of their signals (<b>E</b>,<b>H</b>). Statistical significance levels are indicated as: * (<span class="html-italic">p</span> &lt; 0.05); *** (<span class="html-italic">p</span> &lt; 0.001); **** (<span class="html-italic">p</span> &lt; 0.0001).</p> Full article ">Figure 6
<p>Flow cytometry studies of cellular uptake behavior of C6-Man-SLNs on Raw 264.7 cells with or without mannose preincubation (<b>A</b>) and their quantitative analyses (<b>B</b>). Statistical significance levels are indicated as: * (<span class="html-italic">p</span> &lt; 0.05); ** (<span class="html-italic">p</span> &lt; 0.01).</p> Full article ">Figure 7
<p>Cytotoxic potentials of Rif solution and different formulations on Raw 264.7 cells (<b>A</b>) and A549 cells (<b>B</b>) after 24 h of incubation (<span class="html-italic">n</span> = 5, Mean ± SD).</p> Full article ">
13 pages, 3196 KiB  
Article
Utilization and Evaluation of Rice Bran and Rice Bran Wax as a Tablet Lubricant
by Ornanong S. Kittipongpatana, Karnkamol Trisopon, Phanphen Wattanaarsakit and Nisit Kittipongpatana
Pharmaceutics 2024, 16(3), 428; https://doi.org/10.3390/pharmaceutics16030428 - 20 Mar 2024
Cited by 1 | Viewed by 2128
Abstract
The rice bran and rice bran wax of the KJ CMU107 rice strain were investigated as potential tablet lubricants in a directly compressed tablet formulation. Stabilized full-fatted rice bran (sFFRB), stabilized defatted rice bran (sDFRB), and rice bran wax (RBW) extracted and purified [...] Read more.
The rice bran and rice bran wax of the KJ CMU107 rice strain were investigated as potential tablet lubricants in a directly compressed tablet formulation. Stabilized full-fatted rice bran (sFFRB), stabilized defatted rice bran (sDFRB), and rice bran wax (RBW) extracted and purified from crude rice bran oil (cRBO) were tested. Two commercial lubricants, including magnesium stearate (MGS) and hydrogenated cottonseed oil (HVO), were employed as the standards in the formulated mixtures, which contained spray-dried rice starch (SDRS) as a diluent. The tableting was carried out for each formulation, and the obtained tablets were physically and mechanically evaluated. Among the parameters investigated were the general appearance, ejection force, weight variation, hardness, friability, and disintegration time. The powder flow was also determined for each formulation. The results showed that the tablet ejection forces for all the lubricated formulations (58–259 N) were significantly lower than that of the non-lubricated control formulation (349 N). The use of sFFRB as a lubricant at 0.5–2.0% w/w could lower the ejection force up to 78%, but the hardness reduced so drastically that the formulations failed the friability test due to the chipping of the tablets’ edges. Moreover, sDFRB performed significantly better as the use at 0.5–1.0% w/w in the formulation helped to lower the ejection forces by up to 80% while maintaining the changes in the tablet hardness within 10%. RBW functioned effectively as a tablet lubricant at a concentration of 0.5% w/w, yielding tablets with good strength comparable to standard HVO lubricant while helping to reduce the ejection force by 82%. In formulations with good lubrication, i.e., friability < 1%, the powder flow was improved, and the tablet disintegration times were within the same range as the control and HVO formulations. In conclusion, sDFRB displayed a lubricant property at concentrations between 0.5 and 1.0% w/w, with slightly negative effects on the tablet hardness. RBW from KJ CMU107 rice was an effective tablet lubricant at 0.5% w/w, with no effect on tablet hardness. Both materials can be further developed for use as commercial lubricants in direct compression. Full article
(This article belongs to the Special Issue Excipients Used in Pharmaceutical Dosage Forms)
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<p>(<b>A</b>) Full-fatted rice bran (FFRB); (<b>B</b>) cold-pressed rice bran (CPRB); (<b>C</b>) crude rice bran oil (cRBO); (<b>D</b>) defatted rice bran (DFRB).</p> Full article ">Figure 2
<p>Crude RBW isolated from rice bran (<b>left</b>) and after purification (<b>right</b>).</p> Full article ">Figure 3
<p>SEM images (3000×) of (<b>A</b>) full-fatted rice bran (FFRB); (<b>B</b>) KJ CMU107 rice bran wax (RBW); (<b>C</b>) commercial rice bran wax (NatureSoft 860 GMP).</p> Full article ">Figure 4
<p>Tabletability profile of the materials in the tablet formulations.</p> Full article ">Figure 5
<p>Macroscopic (<b>left</b>) and microscopic (<b>right</b>) appearances and remarks of the tablet formulations containing different lubricants. (<b>A</b>) Control (no lubricant); (<b>B</b>) 0.5% MGS; (<b>C</b>) 1.0% sFFRB; (<b>D</b>) 1.0% sDFRB; (<b>E</b>) 0.5% HVO; (<b>F</b>) 0.5% RBW.</p> Full article ">
24 pages, 3524 KiB  
Article
NLC-Based Sunscreen Formulations with Optimized Proportion of Encapsulated and Free Filters Exhibit Enhanced UVA and UVB Photoprotection
by Margarete M. de Araújo, Andressa C. Schneid, Mariana S. Oliveira, Samuel V. Mussi, Miller N. de Freitas, Flávia C. Carvalho, Edson A. Bernes Junior, Renato Faro and Hatylas Azevedo
Pharmaceutics 2024, 16(3), 427; https://doi.org/10.3390/pharmaceutics16030427 - 20 Mar 2024
Cited by 5 | Viewed by 3192
Abstract
The topical use of sunscreens is recommended for avoiding the damaging effects of UV radiation. However, improvements are still needed in the existing products to enhance their photoprotection effectiveness and safety. This involves minimizing the use of chemical UV filters while providing enhanced [...] Read more.
The topical use of sunscreens is recommended for avoiding the damaging effects of UV radiation. However, improvements are still needed in the existing products to enhance their photoprotection effectiveness and safety. This involves minimizing the use of chemical UV filters while providing enhanced and prolonged photoprotection. This work investigated novel sunscreen formulations and their UV protection effects by encapsulating Uvinul® A, Tinosorb® S, and Uvinul® T150 into nanostructured lipid carriers (NLCs) based on bacuri butter and raspberry seed oil. First, the impact of critical formulation and process parameters on NLCs’ particle size was evaluated using a 22 Face Centered Central Composite Design. Then, formulations were evaluated in terms of critical quality factors, in vitro skin permeation, and in vitro and in vivo photoprotection activities. The developed NLCs-containing formulations exhibited appropriate size (122–135 nm), PdI (<0.3), encapsulation efficiency (>90%), and drug content (>80%), which were preserved for at least 90 days under different stability conditions. Moreover, these NLCs-based formulations had equivalent skin permeation to emulsion-based controls, and the addition of NLCs into sunscreen cream bases in the optimum proportion of 20% (w/w) resulted in enhanced UVA and UVB photoprotection levels, despite a 10% reduction in the total filters content. Altogether, these results describe the application of nanoencapsulated organic UV filters in innovative sunscreen formulations to achieve superior photoprotection and cosmeceutical properties. Full article
(This article belongs to the Special Issue Quality Requirements for Nanomedicines: From Early Development to the Market)
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Graphical abstract

Graphical abstract
Full article ">Figure 1
<p>(<b>a</b>) Scheme describing the interaction between UVR (UVA, UVB, and UVC) and the different layers of the skin structure (stratum corneum, epidermis, and dermis). (<b>b</b>) Depiction of different lipid-based nanoparticles applied to sunscreen formulations.</p> Full article ">Figure 2
<p>Design space (yellow region) of SC-NLC varying (<b>a</b>) Polysorbate 60:Polyglyceryl-3-dioleate ratio (<b>b</b>) and HPH parameters—pressure and time of processing.</p> Full article ">Figure 3
<p>(<b>a</b>) AFM image, where the color scale shows that the brighter spots represent regions that are more exposed (in a higher position), and the darker fraction indicates the sample background. The scale shown in the image as reference is 2 µm. The histogram is resultant from a sum of several AFM images, showing the size frequency (non-normalized) of the optimized SC-NLC; (<b>b</b>) UV-Vis light extinction spectra of the mixtures of Uvinul<sup>®</sup> A Plus, Tinosorb<sup>®</sup> S, and Uvinul<sup>®</sup> T150 free and encapsulated in the SC-NLC, also of the mixture of the SC-NLC unloaded (no filters).</p> Full article ">Figure 4
<p>Evolution over time (in days) of SC-NLC’s (<b>a</b>) size (nm), PdI, (<b>b</b>) zeta potential (mV), and pH during three months under different incubation temperature conditions (4, 25, and 40 °C) and relative humidity of 75% (except for 4 °C). For the plot (<b>a</b>), the bars are referent to the particles’ hydrodynamic diameter (D<sub>H</sub>), and the lines express the PdI trend.</p> Full article ">Figure 5
<p>Percentage of UV filter total load and encapsulation efficiency for Uvinul<sup>®</sup> A Plus, Tinosorb<sup>®</sup> S, and Uvinul<sup>®</sup> T150 at relative humidity (RH) of 75% and temperature of (<b>a</b>) 4 °C (refrigerator stability condition—RH not controlled), (<b>b</b>) 25 °C (long term stability condition), (<b>c</b>) 40 °C (accelerated stability condition). For all graphs the bars are referent to the percentage of filter content found in the SC-NLCs sample, and the lines express the percentual of filters encapsulated into the NLCs.</p> Full article ">Figure 6
<p>Permeation of (<b>a</b>) Uvinul<sup>®</sup> A Plus, (<b>b</b>) Tinosorb<sup>®</sup> S, and (<b>c</b>) Uvinul<sup>®</sup> T150 into the stratum corneum, epidermis, dermis, and receptor liquid. This data set was statistically evaluated by the Kruskal–Wallis test and a significance threshold of less than 0.05.</p> Full article ">Figure 7
<p>Photoprotection responses obtained in vivo tests assessing (<b>a</b>) SPF and (<b>b</b>) UVA-PF<sub>0</sub> of CBI-80%, CBI-80%/Emulsion-20%, and CBI-80%/SC-NLC-20%; (<b>c</b>) SPF and (<b>d</b>) UVA-PF<sub>0</sub> of CBII-80%, CBII-80%/Emulsion-20% and CBII-80%/SC-NLC-20%; and (<b>e</b>) SPF and (<b>f</b>) UVA-PF<sub>0</sub> of CBIII-80%, CBIII-80%/Emulsion-20% and CBIII-80%/SC-NLC-20%. The results of SPF and UVA-PF<sub>0</sub> are represented by their median, quartiles, and upper and bottom limits. The statistical difference was evaluated using the Brown–Forsythe and Welch ANOVA test followed by Dunnett’s T3 multiple comparisons test; it was considered significantly different when <span class="html-italic">p</span> &lt; 0.05, and the symbols *, **, ***, and # indicate significant differences.</p> Full article ">
14 pages, 2287 KiB  
Communication
Safety of Repeated Administration of Xenogeneic Human Apoptotic State (Allocetra-OTS) in Sprague Dawley Rats
by Chen Ankri, Oren Hershkovitz, Liat Hershkovitz, Meital Brami, Ronnie Levy, Hadar Sarig, Einat Souli, Barak Reicher, Veronique Amor-Baroukh, Dror Mevorach and Abraham Nyska
Pharmaceutics 2024, 16(3), 426; https://doi.org/10.3390/pharmaceutics16030426 - 20 Mar 2024
Viewed by 1335
Abstract
Apoptotic cells possess immunomodulatory effects that can be utilized to treat imbalanced immune conditions. Information on the preclinical safety of such treatment is sparse. In this study, the safety of apoptotic cells (Allocetra-OTS) was assessed in a GLP toxicological study on Sprague Dawley [...] Read more.
Apoptotic cells possess immunomodulatory effects that can be utilized to treat imbalanced immune conditions. Information on the preclinical safety of such treatment is sparse. In this study, the safety of apoptotic cells (Allocetra-OTS) was assessed in a GLP toxicological study on Sprague Dawley rats. Three doses of Allocetra-OTS or vehicle were administered intravenously (IV) for 3 consecutive days. Animals in the main study were sacrificed on day 4, while animals from the recovery groups were kept for 14 or 28 days. Allocetra-OTS was well tolerated, and no adverse effects were observed in terms of body weight, clinical signs, food consumption, or ophthalmologic observation. Thus, the No Observed Adverse Effect Level (NOAEL) dose was determined as the highest dose administered. An observed elevation in immune cells was suspected to be due to Allocetra-OTS, similarly to other clinical chemistry parameters; however, it was resolved in the recovery phases. Splenomegaly and dose-related extramedullary hematopoiesis (EMH) in the red pulp were observed, with no adverse events, and were considered to be a normal and expected reaction following the IV administration of cell-based therapies. In conclusion, under the conditions of this study, Allocetra-OTS was concluded to be safe, further supporting its potential candidacy for clinical studies. Full article
(This article belongs to the Special Issue Advanced Pharmaceutical Science and Technology in Israel)
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<p>Apoptosis process analysis in Allocetra-OTS cells following annexin V and propidium iodide (PI) staining. Cells of Allocetra-OTS FDP were stained with annexin V (X axis) and propidium iodide (Y axis) to assess the prevalence of these apoptotic cells immediately following product thawing (representative Allocetra-OTS batch).</p> Full article ">Figure 2
<p>Histopathology analysis of spleen. (<b>A</b>). Histopathology section of the spleen from a rat from the vehicle group (1M). Original magnification ×1. (<b>B</b>) Spleen from the high-dose Allocetra-OTS-treated animal group (4M). Original magnification ×1. (<b>C</b>) Spleen from the vehicle group (1M). Long arrows indicate minimal extramedullary hematopoiesis (EMH) in the red pulp. Original magnification ×10. (<b>D</b>) Spleen from the high-dose Allocetra-OTS group (4M). Short arrows indicate the presence of apoptotic cells. Long arrows indicate EMH. Original magnification ×10. (<b>E</b>) Spleen from the vehicle group (1M). Long arrows indicate EMH in the red pulp. Original magnification ×10. (<b>F</b>) Spleen from the high-dose Allocetra-OTS group (4M). Short arrows indicate the presence of apoptotic cells, and long arrows indicate EMH.</p> Full article ">
3 pages, 128 KiB  
Editorial
Preface to Special Issue: Drug Transporters: Regulation and Roles in Therapeutic Strategies
by Guofeng You
Pharmaceutics 2024, 16(3), 425; https://doi.org/10.3390/pharmaceutics16030425 - 20 Mar 2024
Viewed by 1277
Abstract
Drug transporters are membrane proteins, mediating, across cell membranes, the absorption, distribution, and excretion of a diverse array of endogenous and exogenous substances such as nutrients, metabolites, toxins, and drugs [...] Full article
(This article belongs to the Special Issue Drug Transporters: Regulation and Roles in Therapeutic Strategies)
11 pages, 2714 KiB  
Article
Novel Fluorescent Strategy for Discriminating T and B Lymphocytes Using Transport System
by Heewon Cho, Na-Kyeong Hong and Young-Tae Chang
Pharmaceutics 2024, 16(3), 424; https://doi.org/10.3390/pharmaceutics16030424 - 19 Mar 2024
Cited by 1 | Viewed by 1636
Abstract
Fluorescent bioprobes are invaluable tools for visualizing live cells and deciphering complex biological processes by targeting intracellular biomarkers without disrupting cellular functions. In addition to protein-binding concepts, fluorescent probes utilize various mechanisms, including membrane, metabolism, and gating-oriented strategies. This study introduces a novel [...] Read more.
Fluorescent bioprobes are invaluable tools for visualizing live cells and deciphering complex biological processes by targeting intracellular biomarkers without disrupting cellular functions. In addition to protein-binding concepts, fluorescent probes utilize various mechanisms, including membrane, metabolism, and gating-oriented strategies. This study introduces a novel fluorescent mechanism distinct from existing ways. Here, we developed a B cell selective probe, CDrB, with unique transport mechanisms. Through SLC-CRISPRa screening, we identified two transporters, SLCO1B3 and SLC25A41, by sorting out populations exhibiting higher and lower fluorescence intensities, respectively, demonstrating contrasting activities. We confirmed that SLCO1B3, with comparable expression levels in T and B cells, facilitates the transport of CDrB into cells, while SLC25A41, overexpressed in T lymphocytes, actively exports CDrB. This observation suggests that SLC25A41 plays a crucial role in discriminating between T and B lymphocytes. Furthermore, it reveals the potential for the reversible localization of SLC25A41 to demonstrate its distinct activity. This study is the first report to unveil a novel strategy of SLC by exporting the probe. We anticipate that this research will open up new avenues for developing fluorescent probes. Full article
(This article belongs to the Special Issue Transport and Metabolism of Small-Molecule Drugs, 2nd Edition)
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<p>The selective staining mechanism of fluorescent probes. Fluorescent probes broadly have five strategies. Protein-Oriented Live-cell Distinction (POLD): probes have binding targets, especially proteins, inside cells; Carbohydrate-Oriented Live-cell Distinction (COLD): probes have selectivity to carbohydrates to discriminate specific cell types; Lipid-Oriented Live-cell Distinction (LOLD): compositions of phospholipids and cholesterol located in plasma membrane could be a guide to provide selectivity to probes; Metabolism-Oriented Live-cell Distinction (MOLD): metabolic activities can provide selectivity to probes to be trapped inside specific cell types; Gating-Oriented Live-cell Distinction (GOLD): transporters act as main gates for uptake (SLC; solute carrier family) or export (ABC; ATP-binding cassette) probes. Different expression levels of transporters can provide clues for solving the staining mechanisms of probes. This figure was created with <a href="http://BioRender.com" target="_blank">BioRender.com</a>.</p> Full article ">Figure 2
<p>Development of B cell−selective probe. (<b>A</b>) A schematic view of flow cytometry-based screening. The spleen was firstly dissociated into single cells of splenocytes, and the cells were stained with LC (Luminescent Carbohydrate) compounds. After 1 h, the samples were read via flow cytometry, and extracted values were analyzed using the equation for calculating the stain index. (<b>B</b>) The calculated stain index, which shows separation grades in the splenocytes based on the intensity of LC compounds, was plotted in a one-dimensional graph. (<b>C</b>,<b>D</b>) The CDrB structure is displayed. (<b>E</b>) T cell antibody (CD3) shows a negative correlation with the CDrB signal. (<b>F</b>) The isolated T and B lymphocytes from murine spleen incubated with CDrB, and B cell selectivity is shown. <a href="#pharmaceutics-16-00424-f002" class="html-fig">Figure 2</a>A was created with <a href="http://BioRender.com" target="_blank">BioRender.com</a>.</p> Full article ">Figure 3
<p>Transporter identification and validation through CRISPRa−SLC screening. (<b>A</b>) The schematic process of CRISPRa-SLC screening. CRISPRa-SLC library was stained with CDrB, and both sides of the ~3% brighter and dimmer populations were continuously sorted until enrichment. The enriched cells were analyzed via next generation sequencing (NGS), and the results are displayed as circular graphs. (<b>B</b>) With enriched populations, SLCO1B3- and SLC25A41-cloned cells were acquired compared to control cells. Three groups (the control and SLCO1B3- and SLC25A41-clone groups) were stained by CDrB. Then, images were acquired with a ×40 objective water lens. For these groups, the mRNA expression levels of the target transporters in the cloned cells, (<b>C</b>) slco1b3 and (<b>D</b>) slc25a41, were confirmed. To identify the trends of the expression levels of candidate transporters, murine T and B lymphocytes were isolated from the spleen using magnetic-activated cell sorting (MACS). The analysis results are displayed in (<b>E</b>) slco1b3 and (<b>F</b>) slc25a41. <a href="#pharmaceutics-16-00424-f003" class="html-fig">Figure 3</a>A was created with <a href="http://BioRender.com" target="_blank">BioRender.com</a>.</p> Full article ">Figure 4
<p>Proposed mechanism of CDrB. CDrB is taken up by SLCO1B3 within the cells, expresses in both T and B lymphocytes, and localizes within the mitochondria. However, T cells, which overexpress SLC25A41 compared to B lymphocytes, actively efflux CDrB. Based on this phenomenon, we hypothesize that SLC25A41 exhibits reversible localization to properly expel its substrate, in contrast to the inward positioning of the influx transporter SLCO1B3.</p> Full article ">
16 pages, 3937 KiB  
Review
Alginate and Chitosan-Based Delivery Systems for Improving the Bioavailability and Therapeutic Efficacy of Curcumin
by Anand A. Sable, Amit Kunwar and Atanu Barik
Pharmaceutics 2024, 16(3), 423; https://doi.org/10.3390/pharmaceutics16030423 - 19 Mar 2024
Cited by 1 | Viewed by 2291
Abstract
One of the major challenges in harnessing the therapeutic benefits of curcumin (an active ingredient from turmeric) is its poor bioavailability due to its short biological half-life. In this regard, nanoformulations have shown tremendous hope for improving the pharmacokinetic and therapeutic behavior of [...] Read more.
One of the major challenges in harnessing the therapeutic benefits of curcumin (an active ingredient from turmeric) is its poor bioavailability due to its short biological half-life. In this regard, nanoformulations have shown tremendous hope for improving the pharmacokinetic and therapeutic behavior of curcumin by altering its biological stability and bioavailability. Biopolymers, especially alginate and chitosan, have received special attention as excipients to prepare nanoformulations of curcumin due to their abundant availability, biocompatibility, and amicability to form different types of self-assembled structures and ease of undergoing chemical modifications. However, there are certain challenges, such as poor water solubility under physiological conditions and heterogeneity with regard to molecular weight and large-scale production of well-preserved nanostructures. Substantial advancement has been achieved towards overcoming these challenges by developing newer derivatives through a chemical modifications approach, and this has ascertained the suitability of alginate and chitosan as excipients for drug delivery systems (DDS). The present minireview briefly discusses curcumin and its limitation as a drug molecule, carbohydrates as DDS, and the recent developments related to the alginate and chitosan-based nanoformulations of curcumin. Special emphasis has been given to highlighting the impact of alginate and chitosan-based nanoformulations in improving the therapeutic efficacy and bioavailability of curcumin. Full article
(This article belongs to the Special Issue Curcumin Nanoparticles: Delivery and Therapy)
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<p>Major curcuminoids present in turmeric powder. Reproduced with permission from [<a href="#B3-pharmaceutics-16-00423" class="html-bibr">3</a>] (<span class="html-italic">Food Chem. Toxicol.</span> <b>2022</b>, <span class="html-italic">166</span>, 113254).</p> Full article ">Figure 2
<p>Stages of tumor progression inhibited by curcumin as the associated molecular targets are presented (↑—Upregulation, ↓—Downregulation). Reproduced with permission from [<a href="#B11-pharmaceutics-16-00423" class="html-bibr">11</a>] (<span class="html-italic">Cell. Mol. Life Sci.</span> <b>2008</b>, <span class="html-italic">65</span>, 1631–1652).</p> Full article ">Figure 3
<p>Chemical structure of alginate.</p> Full article ">Figure 4
<p>Representation of the possible interaction mechanism between curcumin and Alg Ald-Gel nanogel, postulated model of nanogel and in vitro curcumin release from Alg Ald-Gel nanogel at pH 5 and pH 7.4. The green box highlights the sites of intermolecular hydrogen bonding between the phenolic OH group in curcumin and free OH goup of Alg Ald-Gel nanogel. Reproduced with permission from [<a href="#B57-pharmaceutics-16-00423" class="html-bibr">57</a>] (<span class="html-italic">Mater. Sci. Eng. C</span> <b>2016</b>, <span class="html-italic">68</span>, 251–257).</p> Full article ">Figure 5
<p>The scheme shows commensal flora triggered targeted release of curcumin from alginate-curcumin micelle for ulcerative colitis treatment. Reproduced with permission from [<a href="#B58-pharmaceutics-16-00423" class="html-bibr">58</a>] (<span class="html-italic">Colloids Surf. B:</span> <b>2021</b>, <span class="html-italic">203</span>, 111756).</p> Full article ">Figure 6
<p>The figure shows the synthetic scheme of Cur-Alg ester, its particle size distribution, and the cleavage and release of curcumin by liver homogenate at pH 8. Reproduced with permission from [<a href="#B59-pharmaceutics-16-00423" class="html-bibr">59</a>] (<span class="html-italic">J. Mol. Struct.</span> <b>2023</b>, <span class="html-italic">1283</span>, 135307).</p> Full article ">Figure 7
<p>(<b>A</b>) Schematic illustration of the structure of bioconjugate AA-CUR and the micelle formed by conjugate AA-CUR (<b>B</b>) Curcumin release profile obtained for AA-CUR/oleic acid mixture at 37 °C, pH = 7.4 (<b>C</b>) Analysis of cytotoxicity of AA-CUR bioconjugate performed using different mouse cancer cell lines by MTT assay. * Statistical significance as compared to control (0 µg/mL of curcumin). Reproduced with permission from [<a href="#B60-pharmaceutics-16-00423" class="html-bibr">60</a>] (<span class="html-italic">Eur. Polym. J.</span> <b>2019</b>, <span class="html-italic">113</span>, 208–219).</p> Full article ">Figure 8
<p>Chemical structure of chitosan.</p> Full article ">Figure 9
<p>The images show FE-SEM image of nanoparticles prepared with chitosan: TPP ratio of 4:1 and drug release profiles of curcumin-loaded CS-NP as affected by cross-linking densities. Reproduced with permission from [<a href="#B74-pharmaceutics-16-00423" class="html-bibr">74</a>] (<span class="html-italic">Pharm. Dev. Technol.</span> <b>2013</b>, 18, 591–599).</p> Full article ">Figure 10
<p>Schematic diagram for the nanoparticle assembly of CENP from EGF-conjugated chitosan, TPP, and curcumin. ENP—curcumin-encapsulated and EGF—conjugated chitosan/TPP nanoparticles; EGF—epidermal growth factor; TEM—transmission electron microscopy; TPP—tripolyphosphate. Reproduced with permission from [<a href="#B80-pharmaceutics-16-00423" class="html-bibr">80</a>] (<span class="html-italic">Int. J. Nanomedicine</span> <b>2018</b>, <span class="html-italic">13</span>, 903–916).</p> Full article ">
14 pages, 4923 KiB  
Article
Intracellular Delivery of Therapeutic Protein via Ultrathin Layered Double Hydroxide Nanosheets
by He Zhang, Anle Ge, Yulin Wang, Boran Xia, Xichu Wang, Zhonghui Zheng, Changsheng Wei, Bo Ma, Lin Zhu, Rose Amal, Sung Lai Jimmy Yun and Zi Gu
Pharmaceutics 2024, 16(3), 422; https://doi.org/10.3390/pharmaceutics16030422 - 19 Mar 2024
Cited by 1 | Viewed by 2062
Abstract
The therapeutic application of biofunctional proteins relies on their intracellular delivery, which is hindered by poor cellular uptake and transport from endosomes to cytoplasm. Herein, we constructed a two-dimensional (2D) ultrathin layered double hydroxide (LDH) nanosheet for the intracellular delivery of a cell-impermeable [...] Read more.
The therapeutic application of biofunctional proteins relies on their intracellular delivery, which is hindered by poor cellular uptake and transport from endosomes to cytoplasm. Herein, we constructed a two-dimensional (2D) ultrathin layered double hydroxide (LDH) nanosheet for the intracellular delivery of a cell-impermeable protein, gelonin, towards efficient and specific cancer treatment. The LDH nanosheet was synthesized via a facile method without using exfoliation agents and showed a high loading capacity of proteins (up to 182%). Using 2D and 3D 4T1 breast cancer cell models, LDH–gelonin demonstrated significantly higher cellular uptake efficiency, favorable endosome escape ability, and deep tumor penetration performance, leading to a higher anticancer efficiency, in comparison to free gelonin. This work provides a promising strategy and a generalized nanoplatform to efficiently deliver biofunctional proteins to unlock their therapeutic potential for cancer treatment. Full article
(This article belongs to the Special Issue Smart Drug Delivery Strategies Based on Porous Materials)
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<p>Schematic graphic of the synthesis and intracellular protein delivery of LDH nanosheets (mass ratio of LDH to gelonin 5:6).</p> Full article ">Figure 2
<p>Physicochemical structures of LDH nanosheets, LDH-BSA, and LDH–gelonin, and protein binding capacity of LDH nanosheets. (<b>a</b>) TEM image of LDH nanosheets. Scale bar = 50 nm. (<b>b</b>) and (<b>c</b>) AFM image and corresponding thickness of LDH nanosheets, respectively. Scale bar = 100 nm. (<b>d</b>) XRD patterns of LDH nanosheets, LDH-BSA, and LDH–gelonin. (<b>e</b>) Adsorption isotherm of BSA on LDH nanosheets fitted in Langmuir model with R<sup>2</sup> being 0.923 and the maximum monolayer adsorption capacity (Q<sub>m</sub>) being 1.82 mg (BSA) per mg LDH. The data (n = 2) are expressed as mean ± SD. (<b>f</b>) Zeta potential (n = 3) and (<b>g</b>) FTIR spectra of LDH nanosheets, LDH-BSA, and LDH–gelonin. (<b>h</b>) TEM images of LDH–gelonin. Scale bar = 50 nm. (<b>i</b>) Size distribution of LDH–gelonin in water, saline, DMEM, or PBS.</p> Full article ">Figure 3
<p>In vitro cell viability studies. (<b>a</b>) Cell viability of 4T1 incubated with PBS, LDH, gelonin, and LDH–gelonin with equivalent gelonin concentrations (0.5–8 µg/mL) for 24 h (n = 3). * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001, **** <span class="html-italic">p</span> &lt; 0.0001. (<b>b</b>) Fluorescence images of live and dead cells for 24 h incubation with PBS, LDH (5 µg/mL), gelonin (6 µg/mL), and LDH–gelonin (11 µg/mL). Green and red fluorescence indicate live and dead cells via Calcein AM and PI staining (scale bar: 50 µm). (<b>c</b>) Apoptosis analysis of 4T1 cells incubated with PBS, LDH (5 µg/mL), gelonin (6 µg/mL), and LDH–gelonin (11 µg/mL) via flow cytometry. (<b>d</b>) Cellular uptake evaluation of 4T1 cells after treating with PBS, gelonin (6 µg/mL), and LDH–gelonin (11 µg/mL) for 4 h.</p> Full article ">Figure 4
<p>Intracellular colocalization analysis of LDH–gelonin and gelonin samples. (<b>a</b>,<b>b</b>) and (<b>d</b>,<b>e</b>) Intracellular colocalization of 4T1 cells incubated with LDH–gelonin-488 and gelonin-488 for 4 h and 24 h, respectively (Blue: DAPI; Green: Alexa-488; Red: Lysotracker. Scale bar = 20 µm). (<b>c</b>) and (<b>f</b>) Pearson’s coefficient of LDH–gelonin-488 and gelonin-488 after 4 h and 24 h incubation with 4T1 cells, respectively (red dots: LDH–gelonin; black triangles: gelonin).</p> Full article ">Figure 5
<p>Tumor penetration ability and antitumor activity of LDH–gelonin nanoparticles evaluated on 3D 4T1 spheroids. (<b>a</b>) Images of 3D spheroids after 12 h of incubation with LDH–gelonin-488 nanoparticles. (<b>b</b>) <span class="html-italic">z</span>-axis depth images from the top to the bottom of the treated spheroid. Scanning interval: 4 μm. Scar bar: 50 μm. (<b>c</b>) Representative brightfield images of 4T1 tumor spheroids. Images acquired with the Olympus CKX41 inverted microscope with UPlanFL N 10x objective. Scale bar = 50 µm. (<b>d</b>) Quantitative analysis of tumor growth inhibition via quantified luminescence intensity obtained from the CellTiter Glo assay (n = 3).</p> Full article ">
12 pages, 1147 KiB  
Article
Formulation and Evaluation of Mucoadhesive Oral Care Gel Containing Kaempferia galanga Extract
by Indah Suasani Wahyuni, Irna Sufiawati, Amira Shafuria, Wipawee Nittayananta and Jutti Levita
Pharmaceutics 2024, 16(3), 421; https://doi.org/10.3390/pharmaceutics16030421 - 19 Mar 2024
Cited by 3 | Viewed by 2444
Abstract
The rhizome of Kaempferia galanga (Zingiberaceae) is extensively used in traditional medicine by utilizing its various biological activities. It has been proven that ethyl-para-methoxycinnamate (EPMC) and other polyphenolic compounds are present in considerable amounts in the ethanolic extract of K. galanga [...] Read more.
The rhizome of Kaempferia galanga (Zingiberaceae) is extensively used in traditional medicine by utilizing its various biological activities. It has been proven that ethyl-para-methoxycinnamate (EPMC) and other polyphenolic compounds are present in considerable amounts in the ethanolic extract of K. galanga rhizome (EKG). Our previous study confirmed that a dose of 0.5–1% of EKG demonstrated anti-inflammatory activity and a wound-healing effect in chemical-induced oral mucosal ulcers of Wistar rats. Currently, there are no reports on the formulation of oral gel containing EKG, thus revealing the potential of EKG to be developed as a herbal oral gel for mucosal ulcers. This study aims to formulate the best mucoadhesive oral care gel containing EKG in terms of physical stability. The presence of EPMC and the total phenols in the best EKG gel were also determined. The results revealed that Carbopol 934 is the best gelling agent for EKG gel preparations as proven by its stability during 14 days of storage. The statistical analysis resulted in a significant difference between the physical stability of the Carbopol 934-based EKG gel preparation compared to three commercial oral care gel products (p < 0.05). RP-HPLC chromatograms indicated that EPMC was identified in Carbopol 934-based gels containing 5% and 10% EKG at 6.056 and 6.146 min, respectively, with polyphenol levels of 1201.2557 mg/kg and 1849.1506 mg/kg, respectively. The hedonic test performed on 30 respondents to measure the degree of consumer acceptance and satisfaction confirmed that 5% EKG gel is the most sensorially accepted by the respondents. Data were analyzed using paired t-tests, one-way ANOVA, and a Kruskal–Wallis test. Taken together, the Carbopol 934-based gel containing 5% EKG could potentially be further developed as a topical anti-oral mucosal ulcer drug for clinical purposes. Full article
(This article belongs to the Special Issue Formulation and Delivery of Traditional Medicines and Natural Products)
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<p>Carbopol 934 (F1 and F2) and Na-CMC (F3) containing EKG in gel formulation, observed on day 0 and day 14.</p> Full article ">Figure 2
<p>Comparison of RP-HPLC chromatograms of standard EPMC (<b>a</b>), F1 (<b>b</b>), and F2 (<b>c</b>).</p> Full article ">Figure 3
<p>Histogram of the hedonic test for F1 and F2.</p> Full article ">
17 pages, 4168 KiB  
Article
Analytical Insights into Protein–Alum Interactions and Their Impact on Conformational Epitope
by Alessio Corrado, Mila Toppazzini, Alessandro Vadi, Carmine Malzone, Rosy Galasso, Alessandro Donati, Riccardo De Ricco and Francesco Berti
Pharmaceutics 2024, 16(3), 420; https://doi.org/10.3390/pharmaceutics16030420 - 19 Mar 2024
Viewed by 1823
Abstract
Several alum-adjuvanted vaccines have been licensed in the past 40 years. Despite its extensive and continuous use, the immune mechanism of action of alum adjuvants is not yet completely understood. Many different variables during the formulation process have been assessed as critical for [...] Read more.
Several alum-adjuvanted vaccines have been licensed in the past 40 years. Despite its extensive and continuous use, the immune mechanism of action of alum adjuvants is not yet completely understood. Many different variables during the formulation process have been assessed as critical for alum-adjuvanted vaccines, although most of them are still not yet fully understood. The absence of a clear understanding of all the possible variables regulating the mechanism of action and the behavior that alum adjuvant imposes on the protein antigen may also be related to analytical challenges. For this reason, there is an urgent need for a fast and simple tool that is possible without a preliminary sample manipulation and is able to control the amount and the degree of antigen adsorption levels and their consistency across different production processes. This work attempts to develop new analytical tools with the aim of directly quantifying and assessing both the content and/or the purity of formulated alum-adsorbed antigens, without any preliminary sample manipulation (e.g., antigen desorption) being reported. In addition, the different confirmation/behavior in terms of the response to specific monoclonal antibodies in the presence of different ratios of alum-OH adsorbent antigens have been investigated. As a proxy to develop new analytical tools, three recombinant protein adsorbed models were used as follows: Neisseria adhesin A (NadA), Neisserial Heparin Binding Antigen (NHBA), and factor H binding protein (fHbp) as antigens, as well as aluminum hydroxide (AH) as an adjuvant system. The selection of the adjuvanted system model was dictated due to the substantial quantity of the literature regarding the protein structure and immunological activities, meaning that they are well characterized, including their adhesion rate to alum. In conclusion, three different analytical tools were explored to quantify, detect, and study the behavior of antigens in the presence of the alum adjuvant. Full article
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<p>Schematic representation of IVRP-Luminex workflow.</p> Full article ">Figure 2
<p>HRF schematic protocol workflow for alum-compound.</p> Full article ">Figure 3
<p>Electropherogram of NHBA with alum at different concentrations: (<b>A</b>): Recombinant protein was diluted at 0.3 mg/mL in ultra-purified water and aluminum hydroxide was added at a proper volume to achieve the desired final concentration of 0.1, 0.3, 0.5 mg/mL. Analytical method was performed with neutral capillary, length tot. 50.2 cm, id. 50 µm; BGE: Tris Acetate 100 mM pH 7.5 + SDS 15 mM; wash at 50 psi for 5 min with water, at 50 psi for 6 min with BGE. Autosampler temperature at 15 °C. Injection by pressure at 0.5 psi for 60.0 s. Separation at 12.0 kV (reverse polarity) + 0.2 psi. (<b>B</b>): Correct area of the NHBA in the presence of alum at increasing concentrations: the graph shows a decrease in corrected area of the protein, but it is not possible to follow the adsorption process on alum.</p> Full article ">Figure 4
<p>Electropherogram profiles of fHbp (blue square); NHBA (green square); NadA (orange square): three proteins were mixed in ultra-pure water to reach a final concentration of 0.1 mg/mL each. Separation method: Bare fused silica capillary, length tot. 70.2 cm, id. 50 µm; BGE: Tris Acetate 50 mM pH 8 + SDS 15 mM; 10 psi 3 min with water, 10 psi 3 min with HCl 0.1 M, 10 psi 4 min NaOH 0.1 M, 10.0 psi BGE. Autosampler temperature at 15 °C. Injection by pressure 0.5 psi 0.15 s. Separation 15.0 kV 40 min. The samples were injected in duplicate (black line first replicate; red line second replicate). Method shows good separation reproducibility and resolution between the three antigens mixed together.</p> Full article ">Figure 5
<p>(<b>A</b>) A.1 The electropherogram profiles on NHBA, with alum at different concentrations (from bottom to the top: no alum, alum 0.3, 0.5, 1, 2, 3 mg/mL), A.2 The trend of corrected areas in function of alum concentration. (<b>B</b>) B.1 The electropherogram profiles on NadA, with alum at different concentrations (from bottom to the top: alum 0.3, 0.5, 1, 2, 3 mg/mL), B.2 The trend of corrected areas in function of alum concentration. (<b>C</b>) C.1 The electropherogram profiles on fHbp, with alum at different concentrations (from bottom to the top: alum 0.3, 0.5, 1, 2, 3 mg/mL), C.2 The trend of corrected areas in function of alum concentration. To evaluate the trend of corrected areas of each antigen, where multiple signals are present, as the value, the sum of all signals observed was considered.</p> Full article ">Figure 6
<p>Representation of relative potency (RP) data obtained at three different time points for multivalent formulations (fHbp; NHBA; NadA) at different alum concentrations. The data were divided for each antigen at the different time points (2 weeks, 2 months, 4 months). The formulations at alum concentrations (2.0, 1.0, 0.5 mg/mL) were tested against the formulation at 3.0 mg/mL. The reference as such has an RP = 1 represented in the graph with a dotted line. Table reports the RP values obtained for each antigen.</p> Full article ">Figure 7
<p>(<b>A</b>) Relative potency (RP) for freshly multivalent formulations (fHbp; NHBA; NadA) at different alum concentrations. The relative potencies for each antigen in the presence of different alum concentrations (3, 2, 1, 0.5 mg/mL) were compared to the RP = 1 reference old multivalent formulate at 3.0 mg/mL. The data shown as the freshly 3.0 mg/mL formulate presents the same potency of the old. Table reports the RP values obtained for each antigen. (<b>B</b>) Relative potency (RP) for freshly multivalent formulations (fHbp; NHBA; NadA) at different alum concentrations. The relative potency for each antigen in the presence of different alum concentrations (2, 1, 0.5 mg/mL) were compared to the RP = 1 reference freshly multivalent formulate at 3.0 mg/mL. Table reports the RP values obtained for each antigen.</p> Full article ">Figure 8
<p>Schematic of an HRF experiment for rate determination. (1) Illustration of a generic protein, where residues on a protein in red are exposed to solvent and more prone to HRF, whereas other residues, in blue, are buried and less exposed due to tight packing and contact formation. (2) Covalent labeling of protein sites by hydroxyl radicals (green dot) that are generated from Fenton chemistry. (3) The enzymatic-digestion-broken protein in small peptide segments, cleaved by a specific protease. (4 and 5) Sequence and site of modified peptides are identified and the amount of modification is quantified based on tandem mass spectroscopy analyses. (6) A characteristic footprinting rate is determined for each peptide/residue segment based on a slope of oxidation rate as a function of exposure time (adapted from [<a href="#B37-pharmaceutics-16-00420" class="html-bibr">37</a>]).</p> Full article ">Figure 9
<p>Comparison of NHBA formulations at different alum concentrations (3.0, 2.0, 1.0, 0.5 mg/mL). (<b>A</b>) Details of the oxidation rate of the three methionine residues 80, 100, and 105 are shown at the top as linear regression plots. (<b>B</b>) The slopes of the oxidation rate of the methionine residues are represented in barrel plots in the lower part, where the numerical values of the linear slopes for the three methionine residues are shown. A higher value corresponds to greater accessibility to the solvent.</p> Full article ">
13 pages, 722 KiB  
Article
The Physicochemical Compatibility of Sildenafil Injection with Parenteral Medications Used in Neonatal Intensive Care Settings
by D. Thisuri N. De Silva, Tobias Strunk, Michael Petrovski, Madhu Page-Sharp, Brioni R. Moore and Kevin T. Batty
Pharmaceutics 2024, 16(3), 419; https://doi.org/10.3390/pharmaceutics16030419 - 18 Mar 2024
Viewed by 2527
Abstract
Sildenafil is used to treat pulmonary hypertension in neonatal intensive care unit (NICU) settings. As multiple intravenous (IV) medications are co-administered in NICU settings, we sought to investigate the physicochemical compatibility of sildenafil with a range of IV drugs. Sildenafil 600 mcg/mL or [...] Read more.
Sildenafil is used to treat pulmonary hypertension in neonatal intensive care unit (NICU) settings. As multiple intravenous (IV) medications are co-administered in NICU settings, we sought to investigate the physicochemical compatibility of sildenafil with a range of IV drugs. Sildenafil 600 mcg/mL or 60 mcg/mL was mixed 1:1 with the secondary drug solution to simulate Y-site co-administration procedures. Physical compatibility was evaluated by visual observation against a black and white background and under polarized light for two hours for changes in colour, precipitation, haze and evolution of gas. Chemical compatibility was determined from sildenafil concentrations, using a validated, stability-indicating high-performance liquid chromatography assay. Sildenafil 600 mcg/mL was physicochemically compatible with 29 of the 45 drugs tested at ‘high-end’ clinical concentrations and physically incompatible with 16 drugs and six ‘2-in-1’ parenteral nutrition solutions. Sildenafil 600 mcg/mL was compatible with lower, clinically relevant concentrations of calcium gluconate, heparin and hydrocortisone. Aciclovir, amoxicillin, ampicillin, ibuprofen lysine, indometacin, phenobarbitone and rifampicin were incompatible with sildenafil 600 mcg/mL, however these IV medications were compatible with sildenafil 60 mcg/mL. Sildenafil 600 mcg/mL and 60 mcg/mL were incompatible with amphotericin, flucloxacillin, furosemide, ibuprofen, meropenem and sodium bicarbonate. Sildenafil compatibility with commonly used syringe filters was also investigated. Sildenafil solution was compatible with nylon syringe filters, however, absorption/adsorption loss occurred with polyethersulfone and cellulose ester filters. Full article
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<p>Recovery (%) of sildenafil 600 mcg/mL injection solution from sterilising filters. Sildenafil concentration was determined from each of four successive millilitres of solution passed through filters (• nylon; ○ polyethersulfone; ▼ mixed cellulose esters; △ inline polyethersulfone; see online <a href="#app1-pharmaceutics-16-00419" class="html-app">Supplementary File Table S5</a> for further details). Data are mean ± SD (<span class="html-italic">n</span> = 3).</p> Full article ">Figure 2
<p>Recovery (%) of sildenafil 60 mcg/mL injection solution from sterilising filters. Sildenafil concentration was determined from each of four successive millilitres of solution passed through filters (• nylon; ○ polyethersulfone; ▼ mixed cellulose esters; △ inline polyethersulfone; see online <a href="#app1-pharmaceutics-16-00419" class="html-app">Supplementary File Table S5</a> for further details). Data are mean ± SD (<span class="html-italic">n</span> = 3).</p> Full article ">
14 pages, 1303 KiB  
Article
Cannabinoid-Induced Inhibition of Morphine Glucuronidation and the Potential for In Vivo Drug–Drug Interactions
by Shelby Coates, Keti Bardhi and Philip Lazarus
Pharmaceutics 2024, 16(3), 418; https://doi.org/10.3390/pharmaceutics16030418 - 18 Mar 2024
Cited by 2 | Viewed by 2809
Abstract
Opioids are commonly prescribed for the treatment of chronic pain. Approximately 50% of adults who are prescribed opioids for pain co-use cannabis with their opioid treatment. Morphine is primarily metabolized by UDP-glucuronosyltransferase (UGT) 2B7 to an inactive metabolite, morphine-3-glucuronide (M3G), and an active [...] Read more.
Opioids are commonly prescribed for the treatment of chronic pain. Approximately 50% of adults who are prescribed opioids for pain co-use cannabis with their opioid treatment. Morphine is primarily metabolized by UDP-glucuronosyltransferase (UGT) 2B7 to an inactive metabolite, morphine-3-glucuronide (M3G), and an active metabolite, morphine-6-glucuronide (M6G). Previous studies have shown that major cannabis constituents including Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD) inhibit major UGT enzymes. To examine whether cannabinoids or their major metabolites inhibit morphine glucuronidation by UGT2B7, in vitro assays and mechanistic static modeling were performed with these cannabinoids and their major metabolites including 11-hydroxy-Δ9-tetrahydrocannabinol (11-OH-THC), 11-nor-9-carboxy-Δ9-tetrahydrocannabinol (11-COOH-THC), 7-hydroxy-cannabidiol (7-OH-CBD), and 7-carboxy-cannabidiol (7-COOH-CBD). In vitro assays with rUGT-overexpressing microsomes and human liver microsomes showed that THC and CBD and their metabolites inhibited UGT2B7-mediated morphine metabolism, with CBD and THC exhibiting the most potent Ki,u values (0.16 µM and 0.37 µM, respectively). Only 7-COOH-CBD exhibited no inhibitory activity against UGT2B7-mediated morphine metabolism. Static mechanistic modeling predicted an in vivo drug–drug interaction between morphine and THC after inhaled cannabis, and between THC, CBD, and 7-OH-CBD after oral consumption of cannabis. These data suggest that the co-use of these agents may lead to adverse drug events in humans. Full article
(This article belongs to the Special Issue Pharmacokinetics and Drug–Drug Interactions of Novel Psychotropic Drugs)
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<p>Hypothesized inhibition of morphine metabolism by cannabinoids.</p> Full article ">Figure 2
<p>Inhibition assay screening of morphine-3-glucuronide and morphine-6-glucuronide formation in rUGT2B7 microsomes (<b>upper panels</b>) and HLM (<b>lower panels</b>).</p> Full article ">Figure 3
<p>Representative <span class="html-italic">IC<sub>50</sub></span> curves in rUGT2B7 microsomes against morphine-3-glucuronide and morphine-6-glucuronide formation.</p> Full article ">Figure 4
<p>Drug interaction risks predicted utilizing static mechanistic models associated with co-use of morphine and increasing clinical doses of THC and CBD. (<b>A</b>), THC and 11-OH-THC after inhalation of THC; (<b>B</b>), THC and 11-OH-THC after oral (po) administration of THC; (<b>C</b>), CBD and 7-OH-CBD after oral (po) administration of CBD. Dashed lines indicate the AUCR cutoff of ≥ 1.25 as recommended by the FDA.</p> Full article ">
17 pages, 1697 KiB  
Article
Medication Lubricants for Oral Delivery of Drugs: Oral Processing Reduces Thickness, Changes Characteristics, and Improves Dissolution Profile
by Marwa A. Malouh, Julie A. Y. Cichero, Yu Sun, Esther T. L. Lau, Lisa M. Nissen and Kathryn J. Steadman
Pharmaceutics 2024, 16(3), 417; https://doi.org/10.3390/pharmaceutics16030417 - 18 Mar 2024
Cited by 1 | Viewed by 1612
Abstract
Swallowing oral solid dosage forms is challenging for those who have medication swallowing difficulties, including patients with dysphagia. One option is to mix the drug (whole or crushed) with a thick vehicle (medication lubricant). Previous in vitro studies consistently suggest that thick vehicles [...] Read more.
Swallowing oral solid dosage forms is challenging for those who have medication swallowing difficulties, including patients with dysphagia. One option is to mix the drug (whole or crushed) with a thick vehicle (medication lubricant). Previous in vitro studies consistently suggest that thick vehicles could impact the dissolution of solid dosage forms, potentially influencing their therapeutic effectiveness, but do not account for changes that happen during oral processing and swallowing. This study aims to investigate the potential impact of medication lubricants on drug release and examine the effect of oral processing. In vitro dissolution of whole and crushed paracetamol tablets mixed with five commercially available medication lubricants (two IDDSI level 2, two IDDSI level 3, and one IDDSI level 4) were tested with and without oral processing; a medication lubricant with/without paracetamol was placed in the mouth (five healthy volunteers), prepared for swallowing, but then expectorated and assessed for physical characteristics and drug release. Medication lubricants, both alone and mixed with crushed paracetamol tablets, showed a significant decrease in viscosity after oral processing. Without oral processing, IDDSI level 3 and 4 lubricants significantly delayed the dissolution of paracetamol tablets. After oral processing, particularly with crushed tablets, there was a substantial increase in the dissolution rate. These findings suggest that dissolution testing overestimates the impact of medication lubricants on drug dissolution. Therefore, using in vitro dissolution tests to predict the dissolution rate of medications mixed with thick vehicles is discouraged. It is essential to consider ways to incorporate the effects of the oral environment and oral processing on thick vehicles used for oral medication administration. Full article
(This article belongs to the Section Pharmaceutical Technology, Manufacturing and Devices)
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<p>Dissolution of paracetamol mixed with MediSpend (<b>a</b>,<b>b</b>), Severo (<b>c</b>,<b>d</b>), Gloup Low Sugar (<b>e</b>,<b>f</b>), Gloup Original (<b>g</b>,<b>h</b>), and Gloup Forte (<b>i</b>,<b>j</b>), with and without oral processing, in simulated gastric fluids using crushed tablets (<b>a</b>,<b>c</b>,<b>e</b>,<b>g</b>,<b>i</b>) and whole tablets (<b>b</b>,<b>d</b>,<b>f</b>,<b>h</b>,<b>j</b>). The bars indicate mean ± se for five replicates.</p> Full article ">Figure 2
<p>Viscosity (Pa·s) of Medispend (<b>A</b>), Severo (<b>B</b>), Gloup Low Sugar (<b>C</b>), Gloup Original (<b>D</b>), and Gloup Forte (<b>E</b>) with and without crushed tablets and/or oral processing were measured at 50 s<sup>−1</sup>. For each medication lubricant analyzed separately using ANOVA with Bonferroni post hoc comparisons, bars with unlike superscript letters above are significantly different (<span class="html-italic">p</span> &lt; 0.05). The data are mean ± se for five replicates.</p> Full article ">
14 pages, 3415 KiB  
Article
Preparation of Nanoparticles Loaded with Membrane-Impermeable Peptide AC3-I and Its Protective Effect on Myocardial Ischemia and Reperfusion
by Yi Liu, Yingyi Niu, Wenjie Zhang, Kaikai Wang, Tianqing Liu and Weizhong Zhu
Pharmaceutics 2024, 16(3), 416; https://doi.org/10.3390/pharmaceutics16030416 - 18 Mar 2024
Cited by 1 | Viewed by 1618
Abstract
Purpose: It is well known that inhibition of Ca2+/calmodulin-dependent protein kinase II (CaMKII) provides cardiac protection in cases of myocardial ischemia–reperfusion injury. However, there are currently no cytoplasm-impermeable drugs that target CaMKII. The aim of this study was to develop curcumin [...] Read more.
Purpose: It is well known that inhibition of Ca2+/calmodulin-dependent protein kinase II (CaMKII) provides cardiac protection in cases of myocardial ischemia–reperfusion injury. However, there are currently no cytoplasm-impermeable drugs that target CaMKII. The aim of this study was to develop curcumin albumin nanoparticles (HSA-CCM NPs) containing AC3-I and investigate their protective effects on hypoxia–reoxygenation (H/R)-induced injuries in adult rat cardiomyocytes and ischemia–reperfusion (I/R) injuries in isolated rat hearts. Methods: HSA-CCM NPs were synthesized using β-ME methods, while the membrane-impermeable peptide AC3-I was covalently linked via a disulfide bond to synthesize AC3-I@HSA-CCM NPs (AC3-I@NPs). Nanoparticle stability and drug release were characterized. To assess the cardiomyocyte uptake of AC3-I@NPs, AC3-I@NPs were incubated with cardiomyocytes under normoxia and hypoxia, respectively. The cardioprotective effect of AC3-I@NPs was determined by using a lactate dehydrogenase kit (LDH) and PI/Hoechst staining. The phosphorylation of phospholamban (p-PLB) was detected by Western blotting in hypoxia–reoxygenation and electric field stimulation models. To further investigate the protective role of AC3-I@NPs against myocardial ischemia–reperfusion injury, we collected coronary effluents and measured creatine kinase (CK) and LDH release in Langendorff rat hearts. Results:AC3-I@NPs were successfully prepared and characterized. Both HSA-CCM NPs and AC3-I@NPs were taken up by cardiomyocytes. AC3-I@NPs protected cardiomyocytes from injury caused by hypoxia–reoxygenation, as demonstrated by decreased cardiomyocyte death and LDH release. AC3-I@NPs reduced p-PLB levels evoked by hypoxia–reoxygenation and electrical field stimulation in adult rat cardiac myocytes. AC3-I@NPs decreased the release of LDH and CK from coronary effluents. Conclusions: AC3-I@NPs showed protective effects against myocardial injuries induced by hypoxia–reoxygenation in cardiomyocytes and ischemia–reperfusion in isolated hearts. Full article
(This article belongs to the Special Issue Biomimetic Nanoparticles for Disease Treatment and Diagnosis)
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Figure 1

Figure 1
<p>Characterization of AC3-I@NPs. (<b>A</b>) TEM image of dry AC3-I@NPs on an aluminum grid. The scale is 200 nanometers. (<b>B</b>) Histogram of size distribution of AC3-I@NPs. (<b>C</b>) UV-Vis absorption spectra of HSA-CCM NPs and AC3-I@NPs. (<b>D</b>) In vitro stability of HSA-CCM NPs and AC3-I@NPs in PBS at 37 °C, pH 7.4. (<b>E</b>) Release of CCM in HSA-CCM NPs and AC3-I@NPs.</p> Full article ">Figure 2
<p>HSA-CCM NPs and AC3-I@NPs were taken up by cardiomyocytes under normoxia and hypoxia. (<b>A</b>,<b>B</b>) Fluorescent images of cellular uptake results. (<b>C</b>) Quantitative measurements of the cellular uptake of HSA-CCM NPs by cardiomyocytes under normoxia or hypoxia for 4 h. (<b>D</b>) Quantitative measurements of the cellular uptake of CCM, HSA-CCM NPs, AC3-I@NPs by cardiomyocytes cultured in normoxia or hypoxia for 4 h. Comparison of normoxia control group, * <span class="html-italic">p</span> &lt; 0.05, *** <span class="html-italic">p</span> &lt; 0.001; comparison of hypoxia control group, <sup>###</sup> <span class="html-italic">p</span> &lt; 0.001; normoxia group compared with hypoxia group, <sup><span>$</span><span>$</span><span>$</span></sup> <span class="html-italic">p</span> &lt; 0.001. Comparison of hypoxia group, *** <span class="html-italic">p</span> &lt; 0.001; comparison of CCM group, <sup><span>$</span><span>$</span><span>$</span></sup> <span class="html-italic">p</span> &lt; 0.001; normoxia group compared with hypoxia group, <sup>###</sup> <span class="html-italic">p</span> &lt; 0.001. <span class="html-italic">n</span> = 3, Mean ± S.E.M.</p> Full article ">Figure 3
<p>AC3-I@NPs had no cytotoxicity to cardiomyocytes. (<b>A</b>) Cytotoxic effects of each group on cardiomyocytes at 10 μM. (<b>B</b>) Toxic effects of different concentrations of AC3-I@NPs on cardiomyocytes. (<b>C</b>) Effect of different concentrations of AIP on myocardial cell toxicity. Compared with the blank control group, there was no significant difference.</p> Full article ">Figure 4
<p>AC3-I@NPs reduced cell death induced by hypoxia–reoxygenation. (<b>A</b>) Plot representative of the PI/Hoechst staining of AC3-I@NPs and AIP. (<b>B</b>) All experimental groups reduced H/R-induced cardiomyocyte death. (<b>C</b>) AC3-I@NPs reduced H/R-induced cardiomyocyte death. (<b>D</b>) AIP reduced H/R-induced cardiomyocyte death. Compared with the normoxia group, <sup>###</sup> <span class="html-italic">p</span> &lt; 0.001; compared with H/R group, * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001; compared with HSA-CCM NPs, <sup><span>$</span><span>$</span></sup> <span class="html-italic">p</span> &lt; 0.01. n = 3, Mean ± S.E.M.</p> Full article ">Figure 4 Cont.
<p>AC3-I@NPs reduced cell death induced by hypoxia–reoxygenation. (<b>A</b>) Plot representative of the PI/Hoechst staining of AC3-I@NPs and AIP. (<b>B</b>) All experimental groups reduced H/R-induced cardiomyocyte death. (<b>C</b>) AC3-I@NPs reduced H/R-induced cardiomyocyte death. (<b>D</b>) AIP reduced H/R-induced cardiomyocyte death. Compared with the normoxia group, <sup>###</sup> <span class="html-italic">p</span> &lt; 0.001; compared with H/R group, * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001; compared with HSA-CCM NPs, <sup><span>$</span><span>$</span></sup> <span class="html-italic">p</span> &lt; 0.01. n = 3, Mean ± S.E.M.</p> Full article ">Figure 5
<p>AC3-I@NPs decreased LDH release after hypoxia–reoxygenation induction. (<b>A</b>) H/R-induced LDH release was decreased in all experimental groups. (<b>B</b>) AC3-I@NPs reduced H/R-induced cardiomyocyte LDH release. (<b>C</b>) AIP reduced H/R-induced LDH release from cardiomyocytes. Compared with normoxia group, <sup>###</sup> <span class="html-italic">p</span> &lt; 0.001; compared with H/R group, * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01; compared with HSA-CCM NPs, <sup><span>$</span></sup> <span class="html-italic">p</span> &lt; 0.05. n = 3, Mean ± S.E.M.</p> Full article ">Figure 6
<p>AC3-I@NPs reduced the expression of P-PLB to reduce CAMKII activity. (<b>A</b>) Western blot analysis for the expression of p-PLB. (<b>B</b>) LDH released from the supernatants of each group. (<b>C</b>) Western blot analysis for the expression of p-PLB under 2 Hz stimulation. Compared with the normoxia group, <sup>###</sup> <span class="html-italic">p</span> &lt; 0.001; compared with the H/R group, * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01; compared with the blank control group, * <span class="html-italic">p</span> &lt; 0.05. n = 3, Mean ± S.E.M.</p> Full article ">Figure 7
<p>AC3-I@NPs alleviated the injuries of cardiac marker enzymes. (<b>A</b>) The change in CK content in the hearts of each group. (<b>B</b>) The change in LDH levels in the hearts of each group. Compared with the normoxia group, <sup>##</sup> <span class="html-italic">p</span> &lt; 0.01, <sup>###</sup> <span class="html-italic">p</span> &lt; 0.001; compared with the I/R Group, * <span class="html-italic">p</span> &lt; 0.05. n = 3, Mean ± S.E.M.</p> Full article ">
20 pages, 3814 KiB  
Article
Trypanocidal and Anti-Inflammatory Effects of Three ent-Kaurane Diterpenoids from Gymnocoronis spilanthoides var. subcordata (Asteraceae)
by Mariana G. Selener, Jimena Borgo, Maria Belen Sarratea, Maria Alicia Delfino, Laura C. Laurella, Natacha Cerny, Jessica Gomez, Mauro Coll, Emilio L. Malchiodi, Augusto E. Bivona, Patricia Barrera, Flavia C. Redko, César A. N. Catalán, Andrés Sánchez Alberti and Valeria P. Sülsen
Pharmaceutics 2024, 16(3), 415; https://doi.org/10.3390/pharmaceutics16030415 - 18 Mar 2024
Viewed by 1873
Abstract
Chagas disease, caused by the protozoan Trypanosoma cruzi, affects 6–7 million people worldwide. The dichloromethane extract obtained from the aerial parts of Gymnocoronis spilanthoides var subcordata showed trypanocidal activity in vitro. The fractionation of the dewaxed organic extract via column chromatography led [...] Read more.
Chagas disease, caused by the protozoan Trypanosoma cruzi, affects 6–7 million people worldwide. The dichloromethane extract obtained from the aerial parts of Gymnocoronis spilanthoides var subcordata showed trypanocidal activity in vitro. The fractionation of the dewaxed organic extract via column chromatography led to the isolation of three diterpenoids: ent-9α,11α-dihydroxy-15-oxo-kaur-16-en-19-oic acid or adenostemmoic acid B, (16R)-ent-11α-hydroxy-15-oxokauran-19-oic acid and ent-11α-hydroxy-15-oxo-kaur-16-en-19-oic acid. These compounds showed IC50 values of 10.6, 15.9 and 4.8 µM against T. cruzi epimastigotes, respectively. When tested against amastigotes, the diterpenoids afforded IC50 values of 6.1, 19.5 and 60.6 µM, respectively. The cytotoxicity of the compounds was tested on mammalian cells using an MTT assay, resulting in CC50s of 321.8, 23.3 and 14.8 µM, respectively. The effect of adenostemmoic acid B on T. cruzi was examined at the ultrastructural level using transmission microscopy. Treatment with 20 μM for 48 h stimulated the formation of abnormal cytosolic membranous structures in the parasite. This compound also showed an anti-inflammatory effect in murine macrophages stimulated with LPS and other TLR agonists. Treatment of macrophages with adenostemmoic acid B was able to reduce TNF secretion and nitric oxide production, while increasing IL-10 production. The combination of adenostemmoic acid B with benznidazole resulted in greater inhibition of NF-kB and a decrease in nitrite concentration. The administration of adenostemmoic acid B to mice infected with trypomastigotes of T. cruzi at the dose of 1 mg/kg/day for five days produced a significant decrease in parasitemia levels and weight loss. Treatment with the association with benznidazole increased the survival time of the animals. In view of these results, adenostemmoic acid B could be considered a promising candidate for further studies in the search for new treatments for Chagas disease. Full article
(This article belongs to the Special Issue Natural Product Pharmaceuticals)
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Graphical abstract

Graphical abstract
Full article ">Figure 1
<p>Chemical structures of phytochemicals isolated from <span class="html-italic">G. spilanthoides</span> var. <span class="html-italic">subcordata</span>. <span class="html-italic">Ent</span>-9α,11α-dihydroxy-15-oxo-kaur-16-en-19-oic acid or adenostemmoic acid B (1), (16<span class="html-italic">R</span>)-<span class="html-italic">ent</span>-11α-hydroxy-15-oxokauran-19-oic acid (2) and <span class="html-italic">ent</span>-11α-hydroxy-15-oxo-kaur-16-en-19-oic acid (3).</p> Full article ">Figure 2
<p>Effect of compounds 1–3 on the epimastigote and amastigote stages of <span class="html-italic">T. cruzi</span>. Every concentration was tested in triplicate to evaluate the inhibition of <span class="html-italic">T. cruzi</span> epimastigotes (<b>A</b>) and amastigotes (<b>B</b>). Results are expressed as the mean ± SEM.</p> Full article ">Figure 3
<p>Cytotoxicity evaluation of the isolated compounds on murine splenocytes. Cells were incubated with increasing concentrations of the compounds, tested in duplicate. (<b>A</b>) Percentage of cell viability in the presence of compounds 1–3. (<b>B</b>) Calculated CC<sub>50</sub> for each compound. Bars represent the means ± SEM.</p> Full article ">Figure 4
<p>(<b>A</b>,<b>B</b>) Untreated parasites displayed typical morphology after a 48 h incubation (<b>C</b>–<b>F</b>) Epimastigotes treated with compound 1 (20 µM, 48 h). Parasites showed the formation of mitochondrial branches (arrowheads) and abnormal cytosolic membranous structures (arrow). N: nucleus, K: kinetoplast, M: mitochondrion, G: Golgi; glycosomes (asterisk).</p> Full article ">Figure 5
<p>(<b>A</b>,<b>B</b>) Non-treated parasites showed typical morphology after a 72 h incubation period. (<b>C</b>,<b>D</b>) Epimastigotes treated with compound 1 (20 µM, 72 h). Abnormal cytosolic membranous structures (white arrowheads) appeared. N: nucleus, K: kinetoplast, M: mitochondrion.</p> Full article ">Figure 6
<p>Effect of compound 1 on RAW-Lucia cells. (<b>A</b>) Luciferase activity after incubation for 24 h with 0, 1 or 10 µg/mL of compound 1 and LPS (0.5 μg/mL) vs. unstimulated control cells. (<b>B</b>) Effect of compound 1 on activation of NF-kB in Raw-Blue cells. Secreted embryonic alkaline phosphatase (SEAP) activity after a 24 h incubation with 0, 1 or 10 µg/mL of compound 1 in treated cells with LPS (0.5 μg/mL) vs. untreated control cells. SEAP activity was determined with the QUANTI-Blue™ reagent Results are expressed as mean ± SEM. * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> ˂ 0.01, *** <span class="html-italic">p</span> &lt; 0.001, two-way ANOVA + Tukey post-test.</p> Full article ">Figure 7
<p>(<b>A</b>) Effect of compound 1 on activation of NF-kB in Raw-Blue cells stimulated with TLR (Toll-like receptors) agonists. SEAP activity was determined 24 h after stimulation with CpG (100 μg/mL), Poly(I:C) (100 μg/mL) or LPS (0.5 μg/mL) in the presence or absence of compound 1 (2.5 µg/mL). Blue bars represent SEAP activity of unstimulated control cells. Results are expressed as the means ± SEM. * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, two-way ANOVA + Sidak post-test. (<b>B</b>) NF-kB activation on Raw-Blue cells stimulated with LPS after combined treatment with compound 1 and Benznidazole (BZ). Raw-blue cells were treated with compound 1 (2.5 µg/mL), BZ (100µM) or both drugs combined either in the presence or absence of LPS (0.5 μg/mL). After a 24 h incubation, SEAP activity was measured. Results are expressed as NF-kB (%) = (Absorbance <sub>treated LPS</sub>-Absorbance <sub>treated DMEM</sub>)/(Absorbance <sub>control LPS</sub>-Absorbance <sub>control DMEM</sub>) × 100. Results are expressed as means ± SEM. ***, <span class="html-italic">p</span> &lt; 0.001. One-way ANOVA+ Tukey post-test.</p> Full article ">Figure 8
<p>Effect of compound 1 on nitric oxide production. (<b>A</b>) Nitrite levels were measured in the supernatants of Raw-Blue cells after stimulation with LPS in the presence of compound 1 using the Griess reagent. Unstimulated cells were used as controls. Results are expressed as means ± SEM. *** <span class="html-italic">p</span> &lt; 0.001, two-way ANOVA + Tukey post-test. (<b>B</b>) Nitrite levels in culture supernatants 24 h post-stimulation with CpG (100 μg/mL), Poly(I:C) (100 μg/mL) and LPS (0.5 μg/mL) in the presence or absence of 2.5 μg/mL of compound 1. Untreated (control group) and unstimulated cells (DMEM group) were included. Results are expressed as means ± SEM. * <span class="html-italic">p</span> &lt; 0.05, *** <span class="html-italic">p</span> &lt; 0.001, two-way ANOVA + Sidak post-test. (<b>C</b>) Raw-Blue cells treated with 2.5 μg/mL of compound 1, 100 μM of BZ, or their combination, in the presence or absence of LPS (0.5 μg/mL). After 24 h post-stimulation, nitric oxide production was determined. Results are expressed as means ± SEM. * <span class="html-italic">p</span> &lt; 0.05, *** <span class="html-italic">p</span> &lt; 0.001, two-way ANOVA + Sidak post-test.</p> Full article ">Figure 9
<p>Effect of compound 1 on the production of cytokines in Raw-Blue macrophages. Cells were cultured in the presence of increasing concentrations of compound 1 (0, 1, or 10 μg/mL) with or without LPS stimulation (0.5 μg/mL). After 24 h, the supernatants were collected. (<b>A</b>) Levels of TNF in culture supernatants evaluated using capture ELISA. The results are expressed as means ± SEM. ** <span class="html-italic">p</span> &lt; 0.01, two-way ANOVA + Tukey post-test. (<b>B</b>) Levels of IL-10 in cultures of Raw-Blue cells treated with increasing concentrations of compound 1 determined by an ELISA assay. ** <span class="html-italic">p</span> &lt; 0.01, one-way ANOVA + Dunnett post-test.</p> Full article ">Figure 10
<p>Efficacy of treatment with compound 1 on a murine model of <span class="html-italic">T. cruzi</span> infection. Female Balb/c mice were infected and then treated with Vehicle (placebo group), compound 1, Benznidazole (BZ) or a combination of Benznidazole and compound 1 for 5 days with a dose of 1 mg/kg/day, intraperitoneally. (<b>A</b>) Parasitemia over time. (<b>B</b>) Area under the parasitemia curve (AUC). Parasitemia was measured weekly via direct counting in a Neubauer chamber. Results are expressed as means ± SEM, ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001. (<b>C</b>) Weight loss. Results are expressed as a percentage of the initial weight at day 0. (<b>D</b>) Kaplan–Meier survival curve for the groups analyzed. The hazard ratio for each group is shown in the table as an indicator of the probability of a near-death event vs. the control group (vehicle).</p> Full article ">
15 pages, 3368 KiB  
Article
Synthesis and Characterization of ZIF-90 Nanoparticles as Potential Brain Cancer Therapy
by Lorenzo Monarca, Francesco Ragonese, Paola Sabbatini, Concetta Caglioti, Matteo Stamegna, Federico Palazzetti, Paolo Sportoletti, Ferdinando Costantino and Bernard Fioretti
Pharmaceutics 2024, 16(3), 414; https://doi.org/10.3390/pharmaceutics16030414 - 18 Mar 2024
Cited by 3 | Viewed by 3093
Abstract
Human glioblastoma is probably the most malignant and aggressive among cerebral tumors, of which it represents approximately 80% of the reported cases, with an overall survival rate that is quite low. Current therapies include surgery, chemotherapy, and radiotherapy, with associated consistent side effects [...] Read more.
Human glioblastoma is probably the most malignant and aggressive among cerebral tumors, of which it represents approximately 80% of the reported cases, with an overall survival rate that is quite low. Current therapies include surgery, chemotherapy, and radiotherapy, with associated consistent side effects and low efficacy. The hardness in reaching the site of action, and overcoming the blood–brain barrier, is a major limitation of pharmacological treatments. In this paper, we report the synthesis and characterization of ZIF-90 (ZIF, Zeolitic Imidazolate Framework) nanoparticles as putative carriers of anticancer drugs to the brain. In particular, we successfully evaluated the biocompatibility of these nanoparticles, their stability in body fluids, and their ability to uptake in U251 human glioblastoma cell lines. Furthermore, we managed to synthesize ZIF-90 particles loaded with berberine, an alkaloid reported as a possible effective adjuvant in the treatment of glioblastoma. These findings could suggest ZIF-90 as a possible new strategy for brain cancer therapy and to study the physiological processes present in the central nervous system. Full article
(This article belongs to the Special Issue Nanomedicines in Cancer Therapy)
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Figure 1
<p>Morphological characterization of ZIF-90. (<b>A</b>) XRPD pattern of ZIF-90. (<b>B</b>) TEM photo of ZIF-90. (<b>C</b>,<b>D</b>) SEM photos of ZIF-90.</p> Full article ">Figure 2
<p>Chemical characterization of ZIF-90. (<b>A</b>) N<sub>2</sub> absorption isotherm at 77 K of ZIF-90 for BET surface area measurement. (<b>B</b>) UV–vis spectrum of ZIF-90.</p> Full article ">Figure 3
<p>The degradation curve of ZIF-90 measured as zinc ion concentration passed through the dialysis membrane over time.</p> Full article ">Figure 4
<p>Dose–response curves of ZIF-90 after 48 h of application of U251 (in black) on HeLa (in red) and HEK293 (in blue) cell lines evaluated based on cell viability via MTT assay.</p> Full article ">Figure 5
<p>The effects of ZIF-90 on hen chick CAM compared with a negative (PBS) and positive (SDS) control. PBS: phosphate-buffered saline; SDS: sodium dodecyl sulfate.</p> Full article ">Figure 6
<p>ZIF-90@Acr characterization. (<b>A</b>) XPRD of ZIF-90 (in black) compared with ZIF-90@Acr (in orange). (<b>B</b>) TG curves of ZIF-90 (in black) compared with ZIF-90@Acr (in orange). The dotted line at 100 °C points to the loss of crystallization water molecules.</p> Full article ">Figure 7
<p>ZIF-90 internalization. (<b>A</b>) Fluorescence analysis of U251 cells after ZIF-90 (in black) or ZIF-90@Acr (in orange) treatment. (<b>B</b>) Example photos taken in Bright Field (BF) and GFP fluorescence channel (merged on the right) of U251 cells treated with ZIF-90@Acr after 1 or 48 h from administration.</p> Full article ">Figure 8
<p>ZIF-90@Berb characterization. XRPD spectra of ZIF-90 (in black) compared with ZIF-90@Berb (in orange).</p> Full article ">Figure 9
<p>HPLC analysis of ZIF-90@Berb particle preparation. (<b>A</b>) Chromatogram of berberine (reference). (<b>B</b>) Chromatogram of ZIF-90. (<b>C</b>) Chromatogram of ZIF-90@Berb.</p> Full article ">
10 pages, 2033 KiB  
Article
Goethite and Hematite Nanoparticles Show Promising Anti-Toxoplasma Properties
by Kosei Ishii, Eiji Akahoshi, Oluyomi Stephen Adeyemi, Hironori Bando, Yasuhiro Fukuda, Tomoyuki Ogawa and Kentaro Kato
Pharmaceutics 2024, 16(3), 413; https://doi.org/10.3390/pharmaceutics16030413 - 18 Mar 2024
Viewed by 1784
Abstract
Toxoplasma gondii is an intracellular parasitic protozoan with a high infection rate in mammals, including humans, and birds. There is no effective vaccine, and treatment relies on antiparasitic drugs. However, existing antiprotozoal drugs have strong side effects and other problems; therefore, new treatment [...] Read more.
Toxoplasma gondii is an intracellular parasitic protozoan with a high infection rate in mammals, including humans, and birds. There is no effective vaccine, and treatment relies on antiparasitic drugs. However, existing antiprotozoal drugs have strong side effects and other problems; therefore, new treatment approaches are needed. Metal nanoparticles have attracted increased interest in the biomedical community in recent years because of their extremely high surface area to volume ratio and their unique reactivity that could be exploited for medicinal purposes. Previously, we confirmed the anti-Toxoplasma effects of gold, silver, and platinum nanoparticles, in a growth inhibition test. Here, we asked whether the anti-Toxoplasma effect could be confirmed with less expensive metal nanoparticles, specifically iron oxide nanoparticles (goethite and hematite). To improve the selective action of the nanoparticles, we modified the surface with l-tryptophan as our previous findings showed that the bio-modification of nanoparticles enhances their selectivity against T. gondii. Fourier-Transform Infrared Spectroscopy (FTIR) analysis confirmed the successful coating of the iron oxide nanoparticles with l-tryptophan. Subsequently, cytotoxicity and growth inhibition assays were performed. L-tryptophan-modified nanoparticles showed superior anti-Toxoplasma action compared to their naked nanoparticle counterparts. L-tryptophan enhanced the selective toxicity of the iron oxide nanoparticles toward T. gondii. The bio-modified nanoparticles did not exhibit detectable host cell toxicity in the effective anti-Toxoplasma doses. To elucidate whether reactive oxygen species contribute to the anti-Toxoplasma action of the bio-modified nanoparticles, we added Trolox antioxidant to the assay medium and found that Trolox appreciably reduced the nanoparticle-induced growth inhibition. Full article
(This article belongs to the Special Issue Anti-parasitic Applications of Nanoparticles)
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Figure 1
<p>In the absence of <span class="html-italic">T. gondii</span> infection, host monolayers were treated with NPs at the effective anti-T. gondii concentration, and cell viability was determined after a 48 h incubation. HFF was seeded at a desired density of 4.0 × 10<sup>4</sup> cells/well. The experiment was conducted three times independently in triplicate. The data shown are the means ± standard deviation (SD).</p> Full article ">Figure 2
<p>Relative number of <span class="html-italic">Toxoplasma</span> parasites after 48 h of infection of HFFs with <span class="html-italic">Toxoplasma</span> and addition of iron oxide nanoparticles (goethite, hematite, and magnetite). Figures are triplicates and averages of three independent runs. Data are means ± standard deviation (SD). Experiments were performed in triplicate and repeated three times independently; ns, not significant at <span class="html-italic">p</span> &gt; 0.05; *, significant at <span class="html-italic">p</span> &lt; 0.05.</p> Full article ">Figure 3
<p>Tryptophan-coated and uncoated iron oxide nanoparticles (goethite and hematite), as well as tryptophan and DMSO, were analyzed by FTIR spectroscopy. The vertical axis shows absorbance, and the horizontal axis shows wavenumber (cm<sup>−1</sup>).</p> Full article ">Figure 4
<p>The cytotoxicity testing of tryptophan-coated iron oxide nanoparticles (goethite and hematite). The maximum concentration was adjusted for both goethite and hematite, and three concentrations were prepared by two-fold dilution. Data are means ± standard deviation (SD).</p> Full article ">Figure 5
<p><span class="html-italic">Toxoplasma</span> growth inhibition using tryptophan-coated iron oxide nanoparticles. Tryptophan-coated iron oxide nanoparticles were added to Toxoplasma-infected cells, and the relative number of <span class="html-italic">Toxoplasma</span> was calculated after 48 h of incubation. (<b>A</b>) Results for tryptophan-coated goethite; (<b>B</b>) results for tryptophan-coated hematite. Data are means ± standard deviation (SD). Experiments were performed in triplicate and repeated three times independently; *, significant at <span class="html-italic">p</span> &lt; 0.05.</p> Full article ">Figure 6
<p>The effect of the antioxidant Trolox on the anti-Toxoplasma effect of iron oxide nanoparticles. Trolox was added after the addition of iron oxide nanoparticles, and the relative survival of <span class="html-italic">Toxoplasma</span> after incubation was calculated. The results of (<b>A</b>) goethite and (<b>B</b>) hematite are shown. Data are means ± standard deviation (SD). Experiments were performed in triplicate and repeated three times independently; ns, not significant at <span class="html-italic">p</span> &gt; 0.05; *, significant at <span class="html-italic">p</span> &lt; 0.05.</p> Full article ">
20 pages, 3201 KiB  
Article
Natural Stabilizers and Nanostructured Lipid Carrier Entrapment for Photosensitive Compounds, Curcumin and Capsaicin
by Wipanan Jandang, Chadarat Ampasavate and Kanokwan Kiattisin
Pharmaceutics 2024, 16(3), 412; https://doi.org/10.3390/pharmaceutics16030412 - 17 Mar 2024
Cited by 3 | Viewed by 1928
Abstract
Capsaicin and curcumin, the active components of chili and turmeric, are prone to instability when exposed to light. Therefore, this research aimed to enhance the photostability of both extracts via the use of antioxidants, natural sunscreen, and nanostructured lipid carriers (NLCs). NLCs were [...] Read more.
Capsaicin and curcumin, the active components of chili and turmeric, are prone to instability when exposed to light. Therefore, this research aimed to enhance the photostability of both extracts via the use of antioxidants, natural sunscreen, and nanostructured lipid carriers (NLCs). NLCs were chosen for this this study due to their advantages in terms of stability, drug loading capacity, occlusive effect, skin penetration, and controlled release. The photostability of each extract and extracts mixed with antioxidants, including grape seed extract, tea extract, and chlorogenic acid, were determined. Chlorogenic acid can enhance the photostability of capsaicin from 6.79 h to 16.50 h, while the photostability of curcumin increased from 9.63 h to 19.25 h. In addition, the use of natural sunscreen (sunflower oil) also increased the photostability of capsaicin and curcumin. The mixed extracts were then loaded into NLCs. The particle size of the formulation was 153.73 nm with a PDI value of 0.25. It exhibited high entrapment efficiency (more than 95%). In addition, it effectively reduced the decomposition of capsaicin and curcumin. Importantly, the natural stabilizers chosen for NLC fabrication significantly improved the photostability of curcumin and capsaicin by 600% and 567% compared to the unstabilized counterparts. This improvement contributes to the sustainability and bioavailability of these compounds in both cosmeceutical and pharmaceutical products. Full article
(This article belongs to the Special Issue Pharmaceutical Solids: Advanced Manufacturing and Characterization)
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<p>Antioxidant activity of the chili extract, turmeric extract, extract mixture, and standards evaluated using the (<b>a</b>) DPPH assay, (<b>b</b>) lipid peroxidation inhibition assay, (<b>c</b>) superoxide radical scavenging assay, and (<b>d</b>) FRAP assay. All tests were performed in triplicate. The <span class="html-italic">p</span> value above the bar graph shows a significant difference between samples at <span class="html-italic">p</span> &lt; 0.05, and NS indicates a nonsignificant difference which was calculated using One-way ANOVA from the SPSS Program.</p> Full article ">Figure 2
<p>DSC thermograms of the chili extract (CH), the turmeric extract (TM), chlorogenic acid (CA), and the chili extract mixed with the turmeric extract and chlorogenic acid (CH + TM + CA).</p> Full article ">Figure 3
<p>Transmission electron microphotographic image of unloaded NLCs (<b>a</b>) and extract-loaded NLCs (<b>b</b>).</p> Full article ">Figure 4
<p>Comparison of the half-lives of capsaicin in the chili extract, the extract mixture, the extract mixture with sunflower oil, and the extract-loaded NLCs (<b>a</b>); the half-lives of curcumin in the turmeric extract, the extract mixture, the extract mixture with sunflower oil, and the extract-loaded NLCs (<b>b</b>) following 8 h of light exposure. The <span class="html-italic">p</span> value below 0.05 above the bar graph indicates a significant difference between each pair of samples, calculated by One-way ANOVA using the SPSS program.</p> Full article ">
26 pages, 1983 KiB  
Review
Fundamentals and Applications of Focused Ultrasound-Assisted Cancer Immune Checkpoint Inhibition for Solid Tumors
by Sepideh Jahangiri and François Yu
Pharmaceutics 2024, 16(3), 411; https://doi.org/10.3390/pharmaceutics16030411 - 16 Mar 2024
Cited by 2 | Viewed by 2695
Abstract
Despite spectacular clinical successes across several cancer types, immune checkpoint inhibition is effective only in subgroups of patients and suffers from significant systemic toxicities, highlighting the need to understand and locally overcome the mechanisms of therapeutic resistance. Similarly to other therapeutics, immunotherapies face [...] Read more.
Despite spectacular clinical successes across several cancer types, immune checkpoint inhibition is effective only in subgroups of patients and suffers from significant systemic toxicities, highlighting the need to understand and locally overcome the mechanisms of therapeutic resistance. Similarly to other therapeutics, immunotherapies face delivery challenges (for example, antibodies need to reach their targets) and immunological barriers that are unique to solid tumors and their microenvironment. Interestingly, focused ultrasound (FUS), with or without microbubbles, which has been shown to enhance gene and drug delivery, notably in oncology, has been recently found to trigger immunological responses. In recent years, there has been a strong emphasis on understanding the biological and immunological effects of FUS for cancer therapy, and FUS is now emerging as an approach that can improve cancer immunotherapy. We herein review: (1) the immunological barriers implicated in ICI resistance; (2) the fundamentals of FUS +/− MB and the current knowledge on leveraging FUS +/− MB bioeffects for improving ICI therapy efficacy; (3) the immune profile of tumor models that have been successfully treated with FUS and ICI; and finally, (4) we discuss the challenges ahead for translating FUS and MB treatments to the clinic, highlighting the exciting perspectives for this new research area. Full article
(This article belongs to the Special Issue Cavitation-Enhanced Drug Delivery and Immunotherapy, 2nd Edition)
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<p>Tumor immune phenotypes defined by the lymphocyte presence, activation, and distribution in the TME. Inflamed tumors are infiltrated by TILs, present a high expression of IFN-γ and PD-L1, and typically respond to ICI. Immune-excluded tumors restrict TILs to the tumor periphery, with a prevalence of myeloid and suppressor cells presenting strong TGF-β signaling with reduced antigen presentation. Immune desert tumors exhibit a complete loss of immune activity (no TILs), and present an altered metabolome, resulting in quick cell proliferation [<a href="#B57-pharmaceutics-16-00411" class="html-bibr">57</a>]. Some tumor examples for each immunophenotype are shown in the figure. <b><span class="html-italic">Acronyms</span></b>: NSCLC: non-small cell lung cancer; CRC: colorectal cancer; HR<sup>+</sup> BC: hormone receptor positive breast cancer.</p> Full article ">Figure 2
<p>An overview of the relationship between I<sub>SPTA</sub>, temperature, pressure, and thermal damage for different FUS modalities. There is minimal thermal damage below the thermal threshold. Thermal damage is strongest for T-HIFU, which has the greatest I<sub>SPTA</sub>. <b><span class="html-italic">Acronyms</span></b>: I<sub>SPTA</sub>: spatial peak temporal average intensity; T-HIFU: thermal HIFU; M-HIFU: mechanical HIFU; pFUS: pulsed focused ultrasound; UTMC: ultrasound-targeted microbubble cavitation.</p> Full article ">Figure 3
<p>Ultrasound parameters and periodic bubble oscillations over several cycles. Basic US parameters that govern bubble behavior are described. Duty cycle is defined as the percentage of ON time divided by the total time (pulse length/pulse repetition period). Pulse repetition frequency (PRF) is the inverse of the pulse repetition period (PRF = 1/PRP). The mechanical index characterizes the extent of cavitation-induced mechanical bioeffects and is calculated as (MI = PNP(MPa)/√F(MHz)). I<sub>SPTA</sub> (spatial peak temporal average intensity) = DC% × (P^2)/(2 × density × sound speed)) describes the time-averaged US power per unit area.</p> Full article ">Figure 4
<p>An overview of the relationship between immune phenotype (x-axis) and FUS modalities (color-coded and localized by pressure on the left y-axis) inducing anti-tumor immune responses in preclinical cancer models. Cancer types are also displayed in the form of pie charts representing their heterogenous clinical presentation as immune inflamed, immune-excluded, and immune desert fractions. Pie charts are localized vertically based on their TMB (right y-axis). <b>Methodology:</b> The immune phenotypes are adapted from [<a href="#B43-pharmaceutics-16-00411" class="html-bibr">43</a>] for melanoma, NSCLC, TNBC, RCC, PDAC, and CRC-MSS. HER2<sup>+</sup>BC is reported to have a similar phenotype to TNBC [<a href="#B167-pharmaceutics-16-00411" class="html-bibr">167</a>,<a href="#B168-pharmaceutics-16-00411" class="html-bibr">168</a>]. The immune phenotypes are determined through TIL infiltration proportions and immune transcriptome profiling in neuroblastoma [<a href="#B169-pharmaceutics-16-00411" class="html-bibr">169</a>], glioma [<a href="#B170-pharmaceutics-16-00411" class="html-bibr">170</a>], gene expression data from the Cancer Genome Atlas for OC [<a href="#B171-pharmaceutics-16-00411" class="html-bibr">171</a>], and TIL infiltration for CRC-MSI [<a href="#B46-pharmaceutics-16-00411" class="html-bibr">46</a>,<a href="#B172-pharmaceutics-16-00411" class="html-bibr">172</a>] and HCC [<a href="#B173-pharmaceutics-16-00411" class="html-bibr">173</a>]. <b>Acronyms:</b> CRC-MSI: colorectal cancer—microsatellite instable; NSCLC: non-small cell lung cancer; RCC: renal cell carcinoma; HER2<sup>+</sup>BC: HER2<sup>+</sup> breast cancer; TNBC: triple-negative breast cancer; CRC-MSS: colorectal cancer—microsatellite stable; PDAC: pancreatic ductal adenocarcinoma; NB: neuroblastoma; OC: ovarian cancer; HCC: hepatocellular carcinoma; TMB: tumor mutational burden; T-HIFU: thermal HIFU; M-HIFU: mechanical HIFU; HT: hyperthermia; pFUS: pulsed FUS; UTMC: ultrasound-targeted microbubble cavitation.</p> Full article ">
18 pages, 1449 KiB  
Review
Role of Biofunctionalized Nanoparticles in Digestive Cancer Vaccine Development
by Razvan Zdrehus, Cristian Delcea and Lucian Mocan
Pharmaceutics 2024, 16(3), 410; https://doi.org/10.3390/pharmaceutics16030410 - 16 Mar 2024
Cited by 1 | Viewed by 2130
Abstract
Nanotechnology has provided an opportunity for unparalleled development of the treatment of various severe diseases. The unique properties of nanoparticles offer a promising strategy for enhancing antitumor immunity by enhancing immunogenicity and presentation of tumor autoantigens for cancer immunotherapy. Polymeric, liposomal, carbon or [...] Read more.
Nanotechnology has provided an opportunity for unparalleled development of the treatment of various severe diseases. The unique properties of nanoparticles offer a promising strategy for enhancing antitumor immunity by enhancing immunogenicity and presentation of tumor autoantigens for cancer immunotherapy. Polymeric, liposomal, carbon or silica-based nanoparticles are among those with major immunomodulatory roles in various cancer treatments. Cancer vaccines, in particular digestive cancer vaccines, have been researched and developed on nanotechnological platforms. Due to their safety, controlled release, targeting of dendritic cells (DCs) and improved antigen uptake, as well as enhanced immunogenicity, nanoparticles have been used as carriers, as adjuvants for increased effect at the tumor level, for their immunomodulating effect, or for targeting the tumor microenvironment, thereby increasing tumor immunogenicity and reducing tumor inflammatory response. This review looks at digestive cancer vaccines developed on nanoparticle platforms and the impact nanoparticles have on the effects of these vaccines. Full article
(This article belongs to the Special Issue Functionalized Nanoparticles in Cancer Therapeutics, 2nd Edition)
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<p>Cancer vaccines can be composed of various platforms to deliver specific tumor antigens. These platforms offer advantages such as simpler manufacturing and flexibility in vaccine delivery. Cell-based vaccines, like dendritic cell (DC) vaccines, allow targeted loading of antigens and manipulation in vivo. However, standardizing manufacturing and quality assessment poses challenges. As our understanding of the immune system grows, there is potential for more efficient and intelligent design of cancer vaccine platforms. These vaccines can be used alone or in combination with other cancer therapies, expanding the scope of cancer immunotherapy as the fourth pillar in oncology, alongside surgery, chemotherapy, and radiation. Reproduced with permission from [<a href="#B4-pharmaceutics-16-00410" class="html-bibr">4</a>].</p> Full article ">Figure 2
<p>The role of DCs in tumor immunity. DCs present antigens to naïve T cells, leading to T-cell activation and transformation into cytotoxic T lymphocytes (CTLs). CTLs then attack tumor cells through direct killing or IFN-γ-dependent pathways. Nanoparticles (NPs) modified with antigens and adjuvants have two main functions: they specifically deliver antigens to DCs, and they promote DC maturation and CTL activation, either by antigen presentation or with the help of adjuvants. This results in the activation and expansion of CD4+ and CD8+ T cells, granting them cytotoxic abilities or helper functions, such as IFN-γ secretion.</p> Full article ">Figure 3
<p>Schematic illustration of the endogenous antigen-carrying nanoparticles (EAC-NPs), EAC-NP formation and the mechanisms of EAC-NP-induced cancer immunotherapy. (<b>A</b>) Schematic illustration of the preparation of HSP70-chaperoned polypeptides HCP+CpG@NPs-CD80 Ab vesicles and induction of T-cell immune responses. (<b>B</b>) Partial magnification of (<b>A</b>). Once phagocytosis occurs, antigen-presenting cells are activated through two signaling pathways: antigen signaling and TLR signaling. After activation, APCs deliver antigen signaling to T lymphocytes, which differentiate into helper T (Th) cells and CTLs and even produce memory T cells. Reproduced with permission from [<a href="#B13-pharmaceutics-16-00410" class="html-bibr">13</a>].</p> Full article ">Figure 4
<p>(<b>a</b>) Fluorescent images of mouse injected with RITC-labeled XL-MSNs subcutaneously on abdomen region, showing targeting of XL-MSNs to the draining lymph node (white dotted circle). (<b>b</b>) OVA-specific and (<b>c</b>) intracellular cytokine-secreting CTLs in the spleens of vaccinated mice measured in flow cytometry (<span class="html-italic">n</span> = 6). Error bars, mean ± s.d. * <span class="html-italic">p</span> &lt; 0.05. (<b>d</b>) Proliferation of CFSE-labeled OVA-specific CD8+ T cells in the lymph node (red line: XL-MSN + OVA + CpG, black line: control), reproduced with permission from [<a href="#B29-pharmaceutics-16-00410" class="html-bibr">29</a>].</p> Full article ">
25 pages, 1517 KiB  
Article
Effects of Combined Treatment with Sodium Dichloroacetate and Sodium Valproate on the Genes in Inflammation- and Immune-Related Pathways in T Lymphocytes from Patients with SARS-CoV-2 Infection with Pneumonia: Sex-Related Differences
by Donatas Stakišaitis, Linas Kapočius, Vacis Tatarūnas, Dovydas Gečys, Auksė Mickienė, Tomas Tamošuitis, Rasa Ugenskienė, Arūnas Vaitkevičius, Ingrida Balnytė and Vaiva Lesauskaitė
Pharmaceutics 2024, 16(3), 409; https://doi.org/10.3390/pharmaceutics16030409 - 16 Mar 2024
Cited by 1 | Viewed by 1877
Abstract
The study presents data on the anti-inflammatory effects of a combination of sodium dichloroacetate and sodium valproate (DCA–VPA) on the expression of inflammation- and immune response-related genes in T lymphocytes of SARS-CoV-2 patients. The study aimed to assess the effects of DCA–VPA on [...] Read more.
The study presents data on the anti-inflammatory effects of a combination of sodium dichloroacetate and sodium valproate (DCA–VPA) on the expression of inflammation- and immune response-related genes in T lymphocytes of SARS-CoV-2 patients. The study aimed to assess the effects of DCA–VPA on the genes of cytokine activity, chemokine-mediated signaling, neutrophil chemotaxis, lymphocyte chemotaxis, T-cell chemotaxis, and regulation of T-cell proliferation pathways. The study included 21 patients with SARS-CoV-2 infection and pneumonia: 9 male patients with a mean age of 68.44 ± 15.32 years and 12 female patients with a mean age of 65.42 ± 15.74 years. They were hospitalized between December 2022 and March 2023. At the time of testing, over 90% of sequences analyzed in Lithuania were found to be of the omicron variant of SARS-CoV-2. The T lymphocytes from patients were treated with 5 mmol DCA and 2 mmol VPA for 24 h in vitro. The effect of the DCA–VPA treatment on gene expression in T lymphocytes was analyzed via gene sequencing. The study shows that DCA–VPA has significant anti-inflammatory effects and apparent sex-related differences. The effect is more potent in T cells from male patients with SARS-CoV-2 infection and pneumonia than in females. Full article
(This article belongs to the Special Issue Antiviral and Antibacterial: Focus on Novel Therapeutic Agents and Drug Delivery Systems)
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<p>Differentially expressed genes in men suffering from SARS-CoV-2 infection with pneumonia: T lymphocytes following DCA–VPA treatment.</p> Full article ">Figure 2
<p>Ridge plot representing the gene set enrichment analysis of inflammation- and immune-related response modulation through DCA–VPA treatment in male patients with SARS-CoV-2 infection and pneumonia.</p> Full article ">Figure 3
<p>Genes whose expression in women’s T lymphocytes was significantly changed by DCA–VPA treatment in patients suffering from SARS-CoV-2 infection with pneumonia.</p> Full article ">Figure 4
<p>Ridge plot representing the gene set enrichment analysis of inflammation- and immune-related response modulation in pathways of interest by DCA–VPA treatment in female patients with SARS-CoV-2 infection and pneumonia.</p> Full article ">Figure 5
<p>Bubble plot representing the impact of DCA–VPA therapy on inflammation- and immune-related gene sets (<b>A</b>) in males with SARS-CoV-2 infection and pneumonia patients, and (<b>B</b>) in females with SARS-CoV-2 infection and pneumonia patients.</p> Full article ">
18 pages, 3548 KiB  
Article
Revolutionizing Three-Dimensional Printing: Enhancing Quality Assurance and Point-of-Care Integration through Instrumentation
by Javier Suárez-González, Eduardo Díaz-Torres, Cecilia N. Monzón-Rodríguez, Ana Santoveña-Estévez and José B. Fariña
Pharmaceutics 2024, 16(3), 408; https://doi.org/10.3390/pharmaceutics16030408 - 16 Mar 2024
Cited by 2 | Viewed by 2050
Abstract
Three-dimensional printing in the field of additive manufacturing shows potential for customized medicines and solving gaps in paediatric formulations. Despite successful clinical trials, 3D printing use in pharmaceutical point-of-care is limited by regulatory loopholes and a lack of Pharmacopoeia guidelines to ensure quality. [...] Read more.
Three-dimensional printing in the field of additive manufacturing shows potential for customized medicines and solving gaps in paediatric formulations. Despite successful clinical trials, 3D printing use in pharmaceutical point-of-care is limited by regulatory loopholes and a lack of Pharmacopoeia guidelines to ensure quality. Semi-solid extrusion is a 3D printing technology that stands out for its versatility, but understanding the fluid dynamics of the semi-solid mass is critical. The aim of this research is to look into the advantages of instrumenting a 3D printer with a semi-solid extrusion motor-driven printhead, which is able to record the printing pressure over time, for in situ characterization of the semi-solid mass and quality evaluation of dosage forms. Four formulations using hydrochlorothiazide as the active pharmaceutical ingredient and several excipients were used. Their flow properties were studied at different printing speeds and temperatures using traditional techniques (rheometer and Texture Analyzer) and the proposed semi-solid extrusion motor-driven printhead incorporated into a printing platform. In addition, the influence of printing speed in the printing process was also evaluated by the study of printing pressure and printlet quality. The results demonstrated the similarities between the use of a Texture Analyzer and the semi-solid extrusion motor-driven. However, the latter enables temperature selection and printing speed in accordance with the printing process which are critical printing parameters. In addition, due to the incorporation of a sensor, it was possible to conclude, for the first time, that there is a link between changes in essential printing parameters like printing speed or formulations and variations in printing pressure and printlet quality attributes such as the energy require to obtain a single dosage unit, weight or diameter. This breakthrough holds a lot of potential for assuring the quality of 3D printing dosage forms and paving the way for their future incorporation into point-of-care settings. Full article
(This article belongs to the Special Issue 3D Printing Technology Applied to the Preparation of Pediatric Orodispersible Dosage Forms)
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<p>Equipment adapted for use in the characterisation of semi-solid mass. (<b>A</b>) Texture analyser; (<b>B</b>) instrumentalized semi-solid extrusion motor-driven printhead.</p> Full article ">Figure 2
<p>Flow test (<b>A</b>), frequency sweep (<b>B</b>), amplitude sweep (<b>C</b>), and creep recovery (<b>D</b>) test for all formulations studied by oscillatory rheometer.</p> Full article ">Figure 3
<p>Characterization of F1 (<b>A</b>), F2 (<b>B</b>), F3 (<b>C</b>), and F4 (<b>D</b>) at different printing speeds using TA and SSE-P.</p> Full article ">Figure 4
<p>Variation in extrusion profile of the formulations when an increase in temperature is applied. (<b>A</b>) F1 and F2 at 25 and 40 °C. (<b>B</b>) F3 and F4 at 25 and 40 °C.</p> Full article ">Figure 5
<p>PAT applied to the 3DP process of the print cartridge. (<b>A</b>,<b>B</b>) Three-dimensional scatter for F1 and 5–30 mm/s. (<b>C</b>) Comparison of printlet for different formulations and printing speed.</p> Full article ">Figure 6
<p>DSC thermal transition curves of pure hydrochlorothiazide and printlet.</p> Full article ">Figure 7
<p>Box plot with outliers for each parameter, formulation, and printing speed studied. (<b>A</b>) Pressure; (<b>B</b>) area under the curve (AUC) (<b>C</b>) weight; (<b>D</b>) diameter; (<b>E</b>) accuracy. ○: Represents cases or rows with values greater than the height of the boxes multiplied by 1.5. *: Represents cases or rows with values greater than the height of the boxes multiplied by three.</p> Full article ">Figure 7 Cont.
<p>Box plot with outliers for each parameter, formulation, and printing speed studied. (<b>A</b>) Pressure; (<b>B</b>) area under the curve (AUC) (<b>C</b>) weight; (<b>D</b>) diameter; (<b>E</b>) accuracy. ○: Represents cases or rows with values greater than the height of the boxes multiplied by 1.5. *: Represents cases or rows with values greater than the height of the boxes multiplied by three.</p> Full article ">Figure 7 Cont.
<p>Box plot with outliers for each parameter, formulation, and printing speed studied. (<b>A</b>) Pressure; (<b>B</b>) area under the curve (AUC) (<b>C</b>) weight; (<b>D</b>) diameter; (<b>E</b>) accuracy. ○: Represents cases or rows with values greater than the height of the boxes multiplied by 1.5. *: Represents cases or rows with values greater than the height of the boxes multiplied by three.</p> Full article ">
21 pages, 1305 KiB  
Review
Nanomedicine for the Treatment of Viral Diseases: Smaller Solution to Bigger Problems
by Suvankar Ghorai, Harshita Shand, Soumendu Patra, Kingshuk Panda, Maria J. Santiago, Md. Sohanur Rahman, Srinivasan Chinnapaiyan and Hoshang J. Unwalla
Pharmaceutics 2024, 16(3), 407; https://doi.org/10.3390/pharmaceutics16030407 - 16 Mar 2024
Cited by 4 | Viewed by 2497
Abstract
The continuous evolution of new viruses poses a danger to world health. Rampant outbreaks may advance to pandemic level, often straining financial and medical resources to breaking point. While vaccination remains the gold standard to prevent viral illnesses, these are mostly prophylactic and [...] Read more.
The continuous evolution of new viruses poses a danger to world health. Rampant outbreaks may advance to pandemic level, often straining financial and medical resources to breaking point. While vaccination remains the gold standard to prevent viral illnesses, these are mostly prophylactic and offer minimal assistance to those who have already developed viral illnesses. Moreover, the timeline to vaccine development and testing can be extensive, leading to a lapse in controlling the spread of viral infection during pandemics. Antiviral therapeutics can provide a temporary fix to tide over the time lag when vaccines are not available during the commencement of a disease outburst. At times, these medications can have negative side effects that outweigh the benefits, and they are not always effective against newly emerging virus strains. Several limitations with conventional antiviral therapies may be addressed by nanotechnology. By using nano delivery vehicles, for instance, the pharmacokinetic profile of antiviral medications can be significantly improved while decreasing systemic toxicity. The virucidal or virus-neutralizing qualities of other special nanomaterials can be exploited. This review focuses on the recent advancements in nanomedicine against RNA viruses, including nano-vaccines and nano-herbal therapeutics. Full article
(This article belongs to the Section Nanomedicine and Nanotechnology)
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<p>Nanoparticle as anti-HIV therapy.</p> Full article ">Figure 2
<p>Polymeric dendrimer targeting HIV.</p> Full article ">Figure 3
<p>Iron oxide NPs with DNAzyme used to target the HCV replication.</p> Full article ">Figure 4
<p>Functionalized nano enzyme used to target hepatitis C virus.</p> Full article ">
16 pages, 3571 KiB  
Article
Useful Role of a New Generation of Dexamethasone, Vitamin E and Human Serum Albumin Microparticles in the Prevention of Excitotoxicity Injury in Retinal Ocular Diseases
by Javier Rodríguez Villanueva, Pedro de la Villa, Rocío Herrero-Vanrell, Irene Bravo-Osuna and Manuel Guzmán-Navarro
Pharmaceutics 2024, 16(3), 406; https://doi.org/10.3390/pharmaceutics16030406 - 15 Mar 2024
Cited by 3 | Viewed by 1458
Abstract
Excitotoxicity has been linked to the pathogenesis of several serious degenerative ocular diseases. Long-term overactivation of the NMDA receptor by glutamate in retinal ganglion cells (RGCs) results in degeneration, apoptosis and loss of function leading to blindness. NMDA receptor antagonists have been proposed [...] Read more.
Excitotoxicity has been linked to the pathogenesis of several serious degenerative ocular diseases. Long-term overactivation of the NMDA receptor by glutamate in retinal ganglion cells (RGCs) results in degeneration, apoptosis and loss of function leading to blindness. NMDA receptor antagonists have been proposed as a pharmacological blockage of glutamate excitotoxicity. However, an inhibition of the pathway activated by glutamate receptors has intolerable side effects. An interesting pharmacological alternative would be the use of antiapoptotic compounds as RGCs’ neuroprotective active substances. Several mechanisms have been proposed to explain neuroprotection, including anti-inflammatory and scavenging activities. Here, the role of dexamethasone in neuroprotection was studied. For this purpose, original controlled release systems composed of microparticles containing dexamethasone with or without vitamin E and human serum albumin (HSA) were designed. The particles were prepared by the solid-in-oil-in-water (S/O/W) emulsion–evaporation technique. After properly characterization of the particles, they were intravitreally injected into an rat model of acute ocular excitotoxicity injury. The functionality of the retina was determined by electroretinography and RGCs were counted after cell immunohistochemistry. These microparticulate systems showed the ability to maintain normal electroretinal activity and promoted significant protection of RGCs. Through this proof of concept, we demonstrated that dexamethasone could be a useful anti-inflammatory agent to avoid the progression of degenerative ocular diseases. Furthermore, when administered in controlled release systems that provide low concentrations during prolonged periods of time, not only can the patient’s comfort be increased but the cytotoxicity of the drugs can also be avoided. Full article
(This article belongs to the Special Issue Suprachoroidal Injection and Beyond: Exploring Advanced Administration Routes for Ocular Diseases)
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<p>Experimental procedure timeline.</p> Full article ">Figure 2
<p>Scheme of retinal areas used to calculate the average cell density. Areas 1, 3, 5 and 7 are 2 mm from the head of the optic nerve, while areas 2, 4, 6 and 8 are in the periphery at 4 mm from the head of the optic nerve.</p> Full article ">Figure 3
<p>Effect of the different treatments on electroretinogram (ERG) wave amplitudes. (<b>A</b>). Representative ERG traces of the rod and the mixed scotopic responses recorded from single animals treated with empty microspheres and loaded microspheres (F1-MSs, F2-MSs and F3-MSs). The rod response was induced by a −2 log cd·s·m<sup>−2</sup> flash; the mixed response was induced by a 1.5 log cd·s·m<sup>−2</sup> flash. Arrows show the measurement for the a-wave and b-wave amplitudes. (<b>B</b>). Histogram representation of the ERG wave amplitudes averaged from 6 animals from each experimental group. Bars correspond to the mean data (mean ± SD) of the b-wave amplitudes measured from the rod response (b-scot), and the a-wave and b-wave amplitudes measured from the mixed response (a-mixed, b-mixed) for the control, NMDA/KA and treated eyes of each experimental group. Statistically significant differences between both eyes are indicated above the bars. n.s.: <span class="html-italic">p</span> &gt; 0.05.</p> Full article ">Figure 4
<p>(<b>A</b>) Density of retinal ganglion cells (RGCs/mm<sup>2</sup>) in each group (* <span class="html-italic">p</span> &lt; 0.01, ** <span class="html-italic">p</span> &lt; 0.05). (<b>B</b>) Density of retinal ganglion cells (RGCs/mm<sup>2</sup>), averaged from each retinal area (1–8, see <a href="#pharmaceutics-16-00406-f002" class="html-fig">Figure 2</a>). The densities for animal groups injected intraocularly with 4 μL of a solution containing NMDA/KA (1 mM and 0.3 mM, respectively) are shown. The histogram bars correspond to the values from the control group (NMDA/KA), group preinjected with 0.1% <span class="html-italic">w/v</span> sodium hyaluronidate (vehicle), Dxm in vehicle and F1-MSs, F2-MSs and F3-MSs in vehicle. The results are displayed as mean ± SD. * <span class="html-italic">p</span> &lt; 0.01.</p> Full article ">Figure 5
<p>Photomicrographs of wholemount retinas stained for Brn3a transcription factor expressed in retinal ganglion cells. The sample images are from animals from each of the experimental groups shown in <a href="#pharmaceutics-16-00406-t002" class="html-table">Table 2</a>. The figure shows the RGC viability in area 3 of the retinas without (control) (<b>A</b>) and after intravitreal injection of 4 μL of a solution containing NMDA/KA (1 mM and 0.3 mM) (<b>B</b>). Images are from the experimental groups pretreated with sodium hyaluronidate 0.1% <span class="html-italic">w</span>/<span class="html-italic">v</span> (vehicle) (<b>C</b>), Dxm in vehicle (<b>D</b>), or F1-MSs (<b>E</b>), F2-MSs (<b>F</b>) or F3-MSs (<b>G</b>) in vehicle, are also shown.</p> Full article ">Figure 6
<p>Effect of the different treatments on retinal photoreceptors and on the inner retina. (<b>A</b>). Photomicrographs of retinal sections stained for cone arrestin (red), cone blue opsin (green) and cell nuclei (blue) from a control eye and eyes injected with Dxm in vehicle, F1-MSs, F2-MSs or F3-MSs in vehicle. No differences among the eyes were observed. (<b>B</b>). Photomicrographs of inner retinal sections stained for the Brn3a transcription factor (green), choline acetyltransferase (red) and cell nuclei (blue) from control and eyes injected with Dxm in vehicle, F1-MSs, F2-MSs or F3-MSs in vehicle. A disorganization of the inner plexiform layer and retinal ganglion cells was observed in the Dxm in vehicle and F3-MSs groups.</p> Full article ">
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