Pharmaceutics

2023

Jump to: 2022, 2021

20 pages, 7221 KiB

Open AccessArticle

Capsules with Ileocolonic-Targeted Release of Vitamin B₂, B₃, and C (ColoVit) Intended for Optimization of Gut Health: Development and Validation of the Production Process

by Aisha A. Ahmed, Antonius T. Otten, Bahez Gareb, Judith E. Huijmans, Anko C. Eissens, Ateequr Rehman, Gerard Dijkstra, Jos G. W. Kosterink, Henderik W. Frijlink and Reinout C. A. Schellekens

Pharmaceutics 2023, 15(5), 1354; https://doi.org/10.3390/pharmaceutics15051354 - 28 Apr 2023

Cited by 1 | Viewed by 1882

Abstract

The ileocolonic-targeted delivery of vitamins can establish beneficial alterations in gut microbial composition. Here, we describe the development of capsules containing riboflavin, nicotinic acid, and ascorbic acid covered with a pH-sensitive coating (ColoVit) to establish site-specific release in the ileocolon. Ingredient properties (particle [...] Read more.

The ileocolonic-targeted delivery of vitamins can establish beneficial alterations in gut microbial composition. Here, we describe the development of capsules containing riboflavin, nicotinic acid, and ascorbic acid covered with a pH-sensitive coating (ColoVit) to establish site-specific release in the ileocolon. Ingredient properties (particle size distribution, morphology) relevant for formulation and product quality were determined. Capsule content and the in vitro release behaviour were determined using a HPLC-method. Uncoated and coated validation batches were produced. Release characteristics were evaluated using a gastro-intestinal simulation system. All capsules met the required specifications. The contents of the ingredients were in the 90.0–120.0% range, and uniformity requirements were met. In the dissolution test a lag-time in drug release of 277–283 min was found, which meets requirements for ileocolonic release. The release itself is immediate as shown by dissolution of the vitamins of more than 75% in 1 h. The production process of the ColoVit formulation was validated and reproducible, it was shown that the vitamin blend was stable during the production process and in the finished coated product. The ColoVit is intended as an innovative treatment approach for beneficial microbiome modulation and optimization of gut health. Full article

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2022

Jump to: 2023, 2021

34 pages, 3856 KiB

Open AccessReview

Liposomal Drug Delivery Systems for Cancer Therapy: The Rotterdam Experience

by Mohamadreza Amin, Ann L. B. Seynhaeve, Majid Sharifi, Mojtaba Falahati and Timo L. M. ten Hagen

Pharmaceutics 2022, 14(10), 2165; https://doi.org/10.3390/pharmaceutics14102165 - 11 Oct 2022

Cited by 15 | Viewed by 3197

Abstract

At the Nanomedicine Innovation Center (NICE) at the Erasmus MC in Rotterdam, we have approached the treatment of cancer by starting with a vision of first establishing a platform that enables us to overcome the low levels of drugs delivered to tumors and [...] Read more.

At the Nanomedicine Innovation Center (NICE) at the Erasmus MC in Rotterdam, we have approached the treatment of cancer by starting with a vision of first establishing a platform that enables us to overcome the low levels of drugs delivered to tumors and the issue of dose-limiting toxicity. Showing that a reduction of the volume of distribution, and a lowering of toxicity and side-effects, accompanied by augmented intratumoral drug delivery, could change outcomes in patients, paved the way to target, not only localized disease, but also systemic and metastasized cancers. In particular, the detailed studies with intravital microscopy we performed at NICE provided us with the necessary insights and affected to a large extent our program on liposome-based cancer therapy. Together with our experience with the loco-regional treatment of cancer, this helped us to develop a program that focused on the subsequent aspects discussed here. We recognized that passive accumulation of nanoparticles was not as effective as previously believed and undertook to improve the local accumulation by changing the tumor pathophysiology and, in particular, the vascular permeability. We added the targeting of liposomes using vascular and tumor directed moieties, to improve cellular drug delivery. To improve payload delivery, we studied the modification of liposomes with phospholipids that help passive drug release and augment cellular accumulation. Second, and importantly, modification of liposomes was undertaken, to enable triggered drug release. The capability for modifying liposomes to respond to a trigger, and the ability to now apply an external trigger (e.g., hyperthermia) and specifically reach the tumor volume, resulted in the current smart drug delivery systems. Our experience at NICE, after a few decades of research on lipid-based nanoparticles, shows that, after the first liposomal formulation registered for clinical application in cancer therapy, further developments quickly followed, while further clinical applications lagged behind. Now we need to focus on and make the next steps towards the clinic, to fulfil the promise that is found there. Full article

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18 pages, 2413 KiB

Open AccessArticle

Dispersing and Sonoporating Biofilm-Associated Bacteria with Sonobactericide

by Kirby R. Lattwein, Inés Beekers, Joop J. P. Kouijzer, Mariël Leon-Grooters, Simone A. G. Langeveld, Tom van Rooij, Antonius F. W. van der Steen, Nico de Jong, Willem J. B. van Wamel and Klazina Kooiman

Pharmaceutics 2022, 14(6), 1164; https://doi.org/10.3390/pharmaceutics14061164 - 30 May 2022

Cited by 5 | Viewed by 2313

Abstract

Bacteria encased in a biofilm poses significant challenges to successful treatment, since both the immune system and antibiotics are ineffective. Sonobactericide, which uses ultrasound and microbubbles, is a potential new strategy for increasing antimicrobial effectiveness or directly killing bacteria. Several studies suggest that [...] Read more.

Bacteria encased in a biofilm poses significant challenges to successful treatment, since both the immune system and antibiotics are ineffective. Sonobactericide, which uses ultrasound and microbubbles, is a potential new strategy for increasing antimicrobial effectiveness or directly killing bacteria. Several studies suggest that sonobactericide can lead to bacterial dispersion or sonoporation (i.e., cell membrane permeabilization); however, real-time observations distinguishing individual bacteria during and directly after insonification are missing. Therefore, in this study, we investigated, in real-time and at high-resolution, the effects of ultrasound-induced microbubble oscillation on Staphylococcus aureus biofilms, without or with an antibiotic (oxacillin, 1 μg/mL). Biofilms were exposed to ultrasound (2 MHz, 100–400 kPa, 100–1000 cycles, every second for 30 s) during time-lapse confocal microscopy recordings of 10 min. Bacterial responses were quantified using post hoc image analysis with particle counting. Bacterial dispersion was observed as the dominant effect over sonoporation, resulting from oscillating microbubbles. Increasing pressure and cycles both led to significantly more dispersion, with the highest pressure leading to the most biofilm removal (up to 83.7%). Antibiotic presence led to more variable treatment responses, yet did not significantly impact the therapeutic efficacy of sonobactericide, suggesting synergism is not an immediate effect. These findings elucidate the direct effects induced by sonobactericide to best utilize its potential as a biofilm treatment strategy. Full article

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17 pages, 5933 KiB

Open AccessArticle

Topography-Mediated Enhancement of Nonviral Gene Delivery in Stem Cells

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

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

Cited by 3 | Viewed by 2456

Abstract

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

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

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19 pages, 2986 KiB

Open AccessReview

Physics of Brain Cancer: Multiscale Alterations of Glioblastoma Cells under Extracellular Matrix Stiffening

by Mohammad Khoonkari, Dong Liang, Marleen Kamperman, Frank A. E. Kruyt and Patrick van Rijn

Pharmaceutics 2022, 14(5), 1031; https://doi.org/10.3390/pharmaceutics14051031 - 10 May 2022

Cited by 26 | Viewed by 6947

Abstract

The biology and physics underlying glioblastoma is not yet completely understood, resulting in the limited efficacy of current clinical therapy. Recent studies have indicated the importance of mechanical stress on the development and malignancy of cancer. Various types of mechanical stress activate adaptive [...] Read more.

The biology and physics underlying glioblastoma is not yet completely understood, resulting in the limited efficacy of current clinical therapy. Recent studies have indicated the importance of mechanical stress on the development and malignancy of cancer. Various types of mechanical stress activate adaptive tumor cell responses that include alterations in the extracellular matrix (ECM) which have an impact on tumor malignancy. In this review, we describe and discuss the current knowledge of the effects of ECM alterations and mechanical stress on GBM aggressiveness. Gradual changes in the brain ECM have been connected to the biological and physical alterations of GBM cells. For example, increased expression of several ECM components such as glycosaminoglycans (GAGs), hyaluronic acid (HA), proteoglycans and fibrous proteins result in stiffening of the brain ECM, which alters inter- and intracellular signaling activity. Several mechanosensing signaling pathways have been identified that orchestrate adaptive responses, such as Hippo/YAP, CD44, and actin skeleton signaling, which remodel the cytoskeleton and affect cellular properties such as cell–cell/ECM interactions, growth, and migration/invasion of GBM cells. In vitro, hydrogels are used as a model to mimic the stiffening of the brain ECM and reconstruct its mechanics, which we also discuss. Overall, we provide an overview of the tumor microenvironmental landscape of GBM with a focus on ECM stiffening and its associated adaptive cellular signaling pathways and their possible therapeutic exploitation. Full article

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15 pages, 2283 KiB

Open AccessArticle

The Effect of Microbubble-Assisted Ultrasound on Molecular Permeability across Cell Barriers

by Charis Rousou, Josanne de Maar, Boning Qiu, Kim van der Wurff-Jacobs, Marika Ruponen, Arto Urtti, Sabrina Oliveira, Chrit Moonen, Gert Storm, Enrico Mastrobattista and Roel Deckers

Pharmaceutics 2022, 14(3), 494; https://doi.org/10.3390/pharmaceutics14030494 - 24 Feb 2022

Cited by 7 | Viewed by 2696

Abstract

The combination of ultrasound and microbubbles (USMB) has been applied to enhance drug permeability across tissue barriers. Most studies focused on only one physicochemical aspect (i.e., molecular weight of the delivered molecule). Using an in vitro epithelial (MDCK II) cell barrier, we examined [...] Read more.

The combination of ultrasound and microbubbles (USMB) has been applied to enhance drug permeability across tissue barriers. Most studies focused on only one physicochemical aspect (i.e., molecular weight of the delivered molecule). Using an in vitro epithelial (MDCK II) cell barrier, we examined the effects of USMB on the permeability of five molecules varying in molecular weight (182 Da to 20 kDa) and hydrophilicity (LogD at pH 7.4 from 1.5 to highly hydrophilic). Treatment of cells with USMB at increasing ultrasound pressures did not have a significant effect on the permeability of small molecules (molecular weight 259 to 376 Da), despite their differences in hydrophilicity (LogD at pH 7.4 from −3.2 to 1.5). The largest molecules (molecular weight 4 and 20 kDa) showed the highest increase in the epithelial permeability (3-7-fold). Simultaneously, USMB enhanced intracellular accumulation of the same molecules. In the case of the clinically relevant anti- C-X-C Chemokine Receptor Type 4 (CXCR4) nanobody (molecular weight 15 kDa), USMB enhanced paracellular permeability by two-fold and increased binding to retinoblastoma cells by five-fold. Consequently, USMB is a potential tool to improve the efficacy and safety of the delivery of drugs to organs protected by tissue barriers, such as the eye and the brain. Full article

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15 pages, 1184 KiB

Open AccessReview

Extracellular Vesicle-Based Hybrid Systems for Advanced Drug Delivery

by Diego A. Rodríguez and Pieter Vader

Pharmaceutics 2022, 14(2), 267; https://doi.org/10.3390/pharmaceutics14020267 - 23 Jan 2022

Cited by 25 | Viewed by 6542

Abstract

The continuous technological advancement of nanomedicine has enabled the development of novel vehicles for the effective delivery of therapeutic substances. Synthetic drug delivery systems are nano-sized carriers made from various materials that can be designed to deliver therapeutic cargoes to cells or tissues. [...] Read more.

The continuous technological advancement of nanomedicine has enabled the development of novel vehicles for the effective delivery of therapeutic substances. Synthetic drug delivery systems are nano-sized carriers made from various materials that can be designed to deliver therapeutic cargoes to cells or tissues. However, rapid clearance by the immune system and the poor targeting profile of synthetic drug delivery systems are examples of the pressing obstacles faced in nanomedicine, which have directed the field toward the development of alternative strategies. Extracellular vesicles (EVs) are nanoscale particles enclosed by a protein-rich lipid bilayer; they are released by cells and are considered to be important mediators of intercellular communication. Owing to their natural composition, EVs have been suggested to exhibit good biocompatibility and to possess homing properties to specific cell types. Combining EVs with synthetic nanoparticles by defined hybridization steps gives rise to a novel potential drug delivery tool, i.e., EV-based hybrid systems. These novel therapeutic vehicles exhibit potential advantageous features as compared to synthetic drug delivery systems such as enhanced cellular uptake and cargo delivery, immuno-evasive properties, capability of crossing biological barriers, and tissue targeting profile. Here, we provide an overview of the various strategies practiced to produce EV-based hybrid systems and elucidate those advantageous features obtained by synthetic drug delivery systems upon hybridization with EVs. Full article

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18 pages, 947 KiB

Open AccessReview

Drug Targeting and Nanomedicine: Lessons Learned from Liver Targeting and Opportunities for Drug Innovation

by Anna Salvati and Klaas Poelstra

Pharmaceutics 2022, 14(1), 217; https://doi.org/10.3390/pharmaceutics14010217 - 17 Jan 2022

Cited by 6 | Viewed by 3853

Abstract

Drug targeting and nanomedicine are different strategies for improving the delivery of drugs to their target. Several antibodies, immuno-drug conjugates and nanomedicines are already approved and used in clinics, demonstrating the potential of such approaches, including the recent examples of the DNA- and [...] Read more.

Drug targeting and nanomedicine are different strategies for improving the delivery of drugs to their target. Several antibodies, immuno-drug conjugates and nanomedicines are already approved and used in clinics, demonstrating the potential of such approaches, including the recent examples of the DNA- and RNA-based vaccines against COVID-19 infections. Nevertheless, targeting remains a major challenge in drug delivery and different aspects of how these objects are processed at organism and cell level still remain unclear, hampering the further development of efficient targeted drugs. In this review, we compare properties and advantages of smaller targeted drug constructs on the one hand, and larger nanomedicines carrying higher drug payload on the other hand. With examples from ongoing research in our Department and experiences from drug delivery to liver fibrosis, we illustrate opportunities in drug targeting and nanomedicine and current challenges that the field needs to address in order to further improve their success. Full article

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18 pages, 3234 KiB

Open AccessArticle

Impact of Formulation Conditions on Lipid Nanoparticle Characteristics and Functional Delivery of CRISPR RNP for Gene Knock-Out and Correction

by Johanna Walther, Danny Wilbie, Vincent S. J. Tissingh, Mert Öktem, Heleen van der Veen, Bo Lou and Enrico Mastrobattista

Pharmaceutics 2022, 14(1), 213; https://doi.org/10.3390/pharmaceutics14010213 - 17 Jan 2022

Cited by 11 | Viewed by 5565

Abstract

The CRISPR-Cas9 system is an emerging therapeutic tool with the potential to correct diverse genetic disorders. However, for gene therapy applications, an efficient delivery vehicle is required, capable of delivering the CRISPR-Cas9 components into the cytosol of the intended target cell population. In [...] Read more.

The CRISPR-Cas9 system is an emerging therapeutic tool with the potential to correct diverse genetic disorders. However, for gene therapy applications, an efficient delivery vehicle is required, capable of delivering the CRISPR-Cas9 components into the cytosol of the intended target cell population. In this study, we optimized the formulation conditions of lipid nanoparticles (LNP) for delivery of ready-made CRISPR-Cas9 ribonucleic protein (RNP). The buffer composition during complexation and relative DOTAP concentrations were varied for LNP encapsulating in-house produced Cas9 RNP alone or Cas9 RNP with additional template DNA for gene correction. The LNP were characterized for size, surface charge, and plasma interaction through asymmetric flow field flow fractionation (AF4). Particles were functionally screened on fluorescent reporter cell lines for gene knock-out and gene correction. This revealed incompatibility of RNP with citrate buffer and PBS. We demonstrated that LNP for gene knock-out did not necessarily require DOTAP, while LNP for gene correction were only active with a low concentration of DOTAP. The AF4 studies additionally revealed that LNP interact with plasma, however, remain stable, whereby HDR template seems to favor stability of LNP. Under optimal formulation conditions, we achieved gene knock-out and gene correction efficiencies as high as 80% and 20%, respectively, at nanomolar concentrations of the CRISPR-Cas9 RNP. Full article

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20 pages, 1619 KiB

Open AccessReview

Receptor Specificity Engineering of TNF Superfamily Ligands

by Fengzhi Suo, Xinyu Zhou, Rita Setroikromo and Wim J. Quax

Pharmaceutics 2022, 14(1), 181; https://doi.org/10.3390/pharmaceutics14010181 - 13 Jan 2022

Cited by 7 | Viewed by 3835

Abstract

The tumor necrosis factor (TNF) ligand family has nine ligands that show promiscuity in binding multiple receptors. As different receptors transduce into diverse pathways, the study on the functional role of natural ligands is very complex. In this review, we discuss the TNF [...] Read more.

The tumor necrosis factor (TNF) ligand family has nine ligands that show promiscuity in binding multiple receptors. As different receptors transduce into diverse pathways, the study on the functional role of natural ligands is very complex. In this review, we discuss the TNF ligands engineering for receptor specificity and summarize the performance of the ligand variants in vivo and in vitro. Those variants have an increased binding affinity to specific receptors to enhance the cell signal conduction and have reduced side effects due to a lowered binding to untargeted receptors. Refining receptor specificity is a promising research strategy for improving the application of multi-receptor ligands. Further, the settled variants also provide experimental guidance for engineering receptor specificity on other proteins with multiple receptors. Full article

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14 pages, 14089 KiB

Open AccessArticle

Simultaneous Exposure of Different Nanoparticles Influences Cell Uptake

by Isa de Boer, Ceri J. Richards and Christoffer Åberg

Pharmaceutics 2022, 14(1), 136; https://doi.org/10.3390/pharmaceutics14010136 - 6 Jan 2022

Cited by 11 | Viewed by 3809

Abstract

Drug delivery using nano-sized carriers holds tremendous potential for curing a range of diseases. The internalisation of nanoparticles by cells, however, remains poorly understood, restricting the possibility for optimising entrance into target cells, avoiding off-target cells and evading clearance. The majority of nanoparticle [...] Read more.

Drug delivery using nano-sized carriers holds tremendous potential for curing a range of diseases. The internalisation of nanoparticles by cells, however, remains poorly understood, restricting the possibility for optimising entrance into target cells, avoiding off-target cells and evading clearance. The majority of nanoparticle cell uptake studies have been performed in the presence of only the particle of interest; here, we instead report measurements of uptake when the cells are exposed to two different types of nanoparticles at the same time. We used carboxylated polystyrene nanoparticles of two different sizes as a model system and exposed them to HeLa cells in the presence of a biomolecular corona. Using flow cytometry, we quantify the uptake at both average and individual cell level. Consistent with previous literature, we show that uptake of the larger particles is impeded in the presence of competing smaller particles and, conversely, that uptake of the smaller particles is promoted by competing larger particles. While the mechanism(s) underlying these observations remain(s) undetermined, we are partly able to restrain the likely possibilities. In the future, these effects could conceivably be used to enhance uptake of nano-sized particles used for drug delivery, by administering two different types of particles at the same time. Full article

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2021

Jump to: 2023, 2022

26 pages, 5000 KiB

Open AccessReview

Polymeric Nanoparticles Properties and Brain Delivery

by Laís Ribovski, Naomi M. Hamelmann and Jos M. J. Paulusse

Pharmaceutics 2021, 13(12), 2045; https://doi.org/10.3390/pharmaceutics13122045 - 30 Nov 2021

Cited by 33 | Viewed by 4195

Abstract

Safe and reliable entry to the brain is essential for successful diagnosis and treatment of diseases, but it still poses major challenges. As a result, many therapeutic approaches to treating disorders associated with the central nervous system (CNS) still only show limited success. [...] Read more.

Safe and reliable entry to the brain is essential for successful diagnosis and treatment of diseases, but it still poses major challenges. As a result, many therapeutic approaches to treating disorders associated with the central nervous system (CNS) still only show limited success. Nano-sized systems are being explored as drug carriers and show great improvements in the delivery of many therapeutics. The systemic delivery of nanoparticles (NPs) or nanocarriers (NCs) to the brain involves reaching the neurovascular unit (NVU), being transported across the blood–brain barrier, (BBB) and accumulating in the brain. Each of these steps can benefit from specifically controlled properties of NPs. Here, we discuss how brain delivery by NPs can benefit from careful design of the NP properties. Properties such as size, charge, shape, and ligand functionalization are commonly addressed in the literature; however, properties such as ligand density, linker length, avidity, protein corona, and stiffness are insufficiently discussed. This is unfortunate since they present great value against multiple barriers encountered by the NPs before reaching the brain, particularly the BBB. We further highlight important examples utilizing targeting ligands and how functionalization parameters, e.g., ligand density and ligand properties, can affect the success of the nano-based delivery system. Full article

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22 pages, 6573 KiB

Open AccessArticle

Internalization and Transport of PEGylated Lipid-Based Mixed Micelles across Caco-2 Cells Mediated by Scavenger Receptor B1

by Xiangjie Su, Mercedes Ramírez-Escudero, Feilong Sun, Joep B. van den Dikkenberg, Mies J. van Steenbergen, Roland J. Pieters, Bert J. C. Janssen, Peter M. van Hasselt, Wim E. Hennink and Cornelus F. van Nostrum

Pharmaceutics 2021, 13(12), 2022; https://doi.org/10.3390/pharmaceutics13122022 - 26 Nov 2021

Cited by 1 | Viewed by 2354

Abstract

The aim of this study was to get insight into the internalization and transport of PEGylat-ed mixed micelles loaded by vitamin K, as mediated by Scavenger Receptor B1 (SR-B1) that is abundantly expressed by intestinal epithelium cells as well as by differentiated Caco-2 [...] Read more.

The aim of this study was to get insight into the internalization and transport of PEGylat-ed mixed micelles loaded by vitamin K, as mediated by Scavenger Receptor B1 (SR-B1) that is abundantly expressed by intestinal epithelium cells as well as by differentiated Caco-2 cells. Inhibition of SR-B1 reduced endocytosis and transport of vitamin-K-loaded 0%, 30% and 50% PEGylated mixed micelles and decreased colocalization of the micelles with SR-B1. Confocal fluorescence microscopy, fluorescence-activated cell sorting (FACS) analysis, and surface plasmon resonance (SPR) were used to study the interaction between the mixed micelles of different compositions (varying vitamin K loading and PEG content) and SR-B1. Interaction of PEGylated micelles was independent of the vitamin K content, indicating that the PEG shell prevented vitamin K exposure at the surface of the micelles and binding with the receptor and that the PEG took over the micelles’ ability to bind to the receptor. Molecular docking calculations corroborated the dual binding of both vita-min K and PEG with the binding domain of SR-B1. In conclusion, the improved colloidal stability of PEGylated mixed micelles did not compromise their cellular uptake and transport due to the affinity of PEG for SR-B1. SR-B1 is able to interact with PEGylated nanoparticles and mediates their subsequent internalization and transport. Full article

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15 pages, 42201 KiB

Open AccessArticle

Aliphatic Quaternary Ammonium Functionalized Nanogels for Gene Delivery

by Huaiying Zhang, Damla Keskin, Willy H. de Haan-Visser, Guangyue Zu, Patrick van Rijn and Inge S. Zuhorn

Pharmaceutics 2021, 13(11), 1964; https://doi.org/10.3390/pharmaceutics13111964 - 19 Nov 2021

Cited by 6 | Viewed by 2797

Abstract

Gene therapy is a promising treatment for hereditary diseases, as well as acquired genetic diseases, including cancer. Facing the complicated physiological and pathological environment in vivo, developing efficient non-viral gene vectors is needed for their clinical application. Here, poly(N-isopropylacrylamide) (p(NIPAM)) nanogels [...] Read more.

Gene therapy is a promising treatment for hereditary diseases, as well as acquired genetic diseases, including cancer. Facing the complicated physiological and pathological environment in vivo, developing efficient non-viral gene vectors is needed for their clinical application. Here, poly(N-isopropylacrylamide) (p(NIPAM)) nanogels are presented with either protonatable tertiary amine groups or permanently charged quaternized ammonium groups to achieve DNA complexation ability. In addition, a quaternary ammonium-functionalized nanogel was further provided with an aliphatic moiety using 1-bromododecane to add a membrane-interacting structure to ultimately facilitate intracellular release of the genetic material. The ability of the tertiary amine-, quaternized ammonium-, and aliphatic quaternized ammonium-functionalized p(NIPAM) nanogels (i.e., NGs, NGs-MI, and NGs-BDD, respectively) to mediate gene transfection was evaluated by fluorescence microscopy and flow cytometry. It is observed that NGs-BDD/pDNA complexes exhibit efficient gene loading, gene protection ability, and intracellular uptake similar to that of NGs-MI/pDNA complexes. However, only the NGs-BDD/pDNA complexes show a notable gene transfer efficiency, which can be ascribed to their ability to mediate DNA escape from endosomes. We conclude that NGs-BDD displays a cationic lipid-like behavior that facilitates endosomal escape by perturbing the endosomal/lysosomal membrane. These findings demonstrate that the presence of aliphatic chains within the nanogel is instrumental in accomplishing gene delivery, which provides a rationale for the further development of nanogel-based gene delivery systems. Full article

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17 pages, 3267 KiB

Open AccessArticle

Retinoic Acid-Containing Liposomes for the Induction of Antigen-Specific Regulatory T Cells as a Treatment for Autoimmune Diseases

by Daniëlle ter Braake, Naomi Benne, Chun Yin Jerry Lau, Enrico Mastrobattista and Femke Broere

Pharmaceutics 2021, 13(11), 1949; https://doi.org/10.3390/pharmaceutics13111949 - 17 Nov 2021

Cited by 10 | Viewed by 3698

Abstract

The current treatment of autoimmune and chronic inflammatory diseases entails systemic immune suppression, which is associated with increased susceptibility to infections. To restore immune tolerance and reduce systemic side effects, a targeted approach using tolerogenic dendritic cells (tolDCs) is being explored. tolDCs are [...] Read more.

The current treatment of autoimmune and chronic inflammatory diseases entails systemic immune suppression, which is associated with increased susceptibility to infections. To restore immune tolerance and reduce systemic side effects, a targeted approach using tolerogenic dendritic cells (tolDCs) is being explored. tolDCs are characterized by the expression of CD11c, the major histocompatibility complex (MHC)II and low levels of co-stimulatory molecules CD40 and CD86. In this study, tolDCs were generated using a human-proteoglycan-derived peptide (hPG) and all-trans retinoic acid (RA). RA-tolDCs not only display a tolerogenic phenotype but also can induce an antigen-specific regulatory T cell (Treg) response in vitro. However, further analysis showed that RA-tolDCs make up a heterogeneous population of DCs, with only a small proportion being antigen-associated tolDCs. To increase the homogeneity of this population, 1,2-distearoyl-sn-glycero-3-phosphoglycerol (DSPG)-containing liposomes were used to encapsulate the relevant antigen together with RA. These liposomes greatly enhanced the proportion of antigen-associated tolDCs in culture. In addition, in mice, we showed that the liposomal co-delivery of antigen and RA can be a more targeted approach to induce antigen-specific tolerance compared to the injection of RA-tolDCs, and that these liposomes can stimulate the generation of antigen-specific Tregs. This work highlights the importance of the co-delivery of an antigen and immunomodulator to minimize off-target effects and systemic side effects and provides new insights in the use of RA for antigen-specific immunotherapy for autoimmune and chronic inflammatory diseases. Full article

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13 pages, 12566 KiB

Open AccessArticle

Mimicking the Biology of Engineered Protein and mRNA Nanoparticle Delivery Using a Versatile Microfluidic Platform

by Valentina Palacio-Castañeda, Rik Oude Egberink, Arbaaz Sait, Lea Andrée, Benedetta Maria Sala, Negar Hassani Besheli, Egbert Oosterwijk, Johan Nilvebrant, Sander C. G. Leeuwenburgh, Roland Brock and Wouter P. R. Verdurmen

Pharmaceutics 2021, 13(11), 1944; https://doi.org/10.3390/pharmaceutics13111944 - 17 Nov 2021

Cited by 6 | Viewed by 3447

Abstract

To investigate the delivery of next-generation macromolecular drugs, such as engineered proteins and mRNA-containing nanoparticles, there is an increasing push towards the use of physiologically relevant disease models that incorporate human cells and do not face ethical dilemmas associated with animal use. Here, [...] Read more.

To investigate the delivery of next-generation macromolecular drugs, such as engineered proteins and mRNA-containing nanoparticles, there is an increasing push towards the use of physiologically relevant disease models that incorporate human cells and do not face ethical dilemmas associated with animal use. Here, we illustrate the versatility and ease of use of a microfluidic platform for studying drug delivery using high-resolution microscopy in 3D. Using this microfluidic platform, we successfully demonstrate the specific targeting of carbonic anhydrase IX (CAIX) on cells overexpressing the protein in a tumor-mimicking chip system using affibodies, with CAIX-negative cells and non-binding affibodies as controls. Furthermore, we demonstrate this system’s feasibility for testing mRNA-containing biomaterials designed to regenerate bone defects. To this end, peptide- and lipid-based mRNA formulations were successfully mixed with colloidal gelatin in microfluidic devices, while translational activity was studied by the expression of a green fluorescent protein. This microfluidic platform enables the testing of mRNA delivery from colloidal biomaterials of relatively high densities, which represents a first important step towards a bone-on-a-chip platform. Collectively, by illustrating the ease of adaptation of our microfluidic platform towards use in distinct applications, we show that our microfluidic chip represents a powerful and flexible way to investigate drug delivery in 3D disease-mimicking culture systems that recapitulate key parameters associated with in vivo drug application. Full article

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16 pages, 4973 KiB

Open AccessArticle

Microfluidic Production of Polymeric Core-Shell Microspheres for the Delayed Pulsatile Release of Bovine Serum Albumin as a Model Antigen

by Renée S. van der Kooij, Rob Steendam, Johan Zuidema, Henderik W. Frijlink and Wouter L. J. Hinrichs

Pharmaceutics 2021, 13(11), 1854; https://doi.org/10.3390/pharmaceutics13111854 - 3 Nov 2021

Cited by 5 | Viewed by 2767

Abstract

For many vaccines, multiple injections are required to confer protective immunity against targeted pathogens. These injections often consist of a primer administration followed by a booster administration of the vaccine a few weeks or months later. A single-injection vaccine formulation that provides for [...] Read more.

For many vaccines, multiple injections are required to confer protective immunity against targeted pathogens. These injections often consist of a primer administration followed by a booster administration of the vaccine a few weeks or months later. A single-injection vaccine formulation that provides for both administrations could greatly improve the convenience and vaccinee’s compliance. In this study, we developed parenterally injectable core-shell microspheres with a delayed pulsatile release profile that could serve as the booster in such a vaccine formulation. These microspheres contained bovine serum albumin (BSA) as the model antigen and poly(dl-lactide-co-glycolide) (PLGA) with various dl-lactide:glycolide monomer ratios as the shell material. Highly monodisperse particles with different particle characteristics were obtained using a microfluidic setup. All formulations exhibited a pulsatile in vitro release of BSA after an adjustable lag time. This lag time increased with the increasing lactide content of the polymer and ranged from 3 to 7 weeks. Shell thickness and bovine serum albumin loading had no effect on the release behavior, which could be ascribed to the degradation mechanism of the polymer, with bulk degradation being the main pathway. Co-injection of the core-shell microspheres together with a solution of the antigen that serves as the primer would allow for the desired biphasic release profile. Altogether, these findings show that injectable core-shell microspheres combined with a primer are a promising alternative for the current multiple-injection vaccines. Full article

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13 pages, 4583 KiB

Open AccessCommunication

Therapeutic Stomatocytes with Aggregation Induced Emission for Intracellular Delivery

by Jingxin Shao, Shoupeng Cao, Hanglong Wu, Loai K. E. A. Abdelmohsen and Jan C. M. van Hest

Pharmaceutics 2021, 13(11), 1833; https://doi.org/10.3390/pharmaceutics13111833 - 2 Nov 2021

Cited by 2 | Viewed by 2486

Abstract

Bowl-shaped biodegradable polymersomes, or stomatocytes, have much potential as drug delivery systems, due to their intriguing properties, such as controllable size, programmable morphology, and versatile cargo encapsulation capability. In this contribution, we developed well-defined therapeutically active stomatocytes with aggregation-induced emission (AIE) features by [...] Read more.

Bowl-shaped biodegradable polymersomes, or stomatocytes, have much potential as drug delivery systems, due to their intriguing properties, such as controllable size, programmable morphology, and versatile cargo encapsulation capability. In this contribution, we developed well-defined therapeutically active stomatocytes with aggregation-induced emission (AIE) features by self-assembly of biodegradable amphiphilic block copolymers, comprising poly(ethylene glycol) (PEG) and AIEgenic poly(trimethylene carbonate) (PTMC) moieties. The presence of the AIEgens endowed the as-prepared stomatocytes with intrinsic fluorescence, which was employed for imaging of cellular uptake of the particles. It simultaneously enabled the photo-mediated generation of reactive oxygen species (ROS) for photodynamic therapy. The potential of the therapeutic stomatocytes as cargo carriers was demonstrated by loading enzymes (catalase and glucose oxidase) in the nanocavity, followed by a cross-linking reaction to achieve stable encapsulation. This provided the particles with a robust motile function, which further strengthened their therapeutic effect. With these unique features, enzyme-loaded AIEgenic stomatocytes are an attractive platform to be exploited in the field of nanomedicine. Full article

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