Publications by authors named "Stefaan C De Smedt"

221 Publications

Hydrogel-Induced Cell Membrane Disruptions Enable Direct Cytosolic Delivery of Membrane-Impermeable Cargo.

Adv Mater 2021 Jun 9:e2008054. Epub 2021 Jun 9.

Ghent Research Group on Nanomedicines, Laboratory of General Biochemistry and Physical Pharmacy, Faculty of Pharmaceutical Sciences, Ghent University, Ottergemsesteenweg 460, Ghent, 9000, Belgium.

Intracellular delivery of membrane-impermeable cargo offers unique opportunities for biological research and the development of cell-based therapies. Despite the breadth of available intracellular delivery tools, existing protocols are often suboptimal and alternative approaches that merge delivery efficiency with both biocompatibility, as well as applicability, remain highly sought after. Here, a comprehensive platform is presented that exploits the unique property of cationic hydrogel nanoparticles to transiently disrupt the plasma membrane of cells, allowing direct cytosolic delivery of uncomplexed membrane-impermeable cargo. Using this platform, which is termed Hydrogel-enabled nanoPoration or HyPore, the delivery of fluorescein isothiocyanate (FITC)-dextran macromolecules in various cancer cell lines and primary bovine corneal epithelial cells is convincingly demonstrated. Of note, HyPore demonstrates efficient FITC-dextran delivery in primary human T cells, outperforming state-of-the-art electroporation-mediated delivery. Moreover, the HyPore platform enables cytosolic delivery of functional proteins, including a histone-binding nanobody as well as the enzymes granzyme A and Cre-recombinase. Finally, HyPore-mediated delivery of the MRI contrast agent gadobutrol in primary human T cells significantly improves their T -weighted MRI signal intensities compared to electroporation. Taken together, HyPore is proposed as a straightforward, highly versatile, and cost-effective technique for high-throughput, ex vivo manipulation of primary cells and cell lines.
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http://dx.doi.org/10.1002/adma.202008054DOI Listing
June 2021

Bubble Forming Films for Spatial Selective Cell Killing.

Adv Mater 2021 May 29:e2008379. Epub 2021 May 29.

Laboratory of General Biochemistry and Physical Pharmacy, Faculty of Pharmaceutical Sciences, Ghent University, Ghent, 9000, Belgium.

Photodynamic and photothermal cell killing at the surface of tissues finds applications in medicine. However, a lack of control over heat dissipation following a treatment with light might damage surrounding tissues. A new strategy to kill cells at the surface of tissues is reported. Polymeric films are designed in which iron oxide nanoparticles are embedded as photosensitizers. Irradiation of the films with pulsed laser light generates water vapor bubbles at the surface of the films. It is found that "bubble-films" can kill cells in close proximity to the films due to mechanical forces which arise when the bubbles collapse. Local irradiation of bubble-films allows for spatial selective single cell killing. As nanosurgery becomes attractive in ophthalmology to remove superficial tumors, bubble-films are applied on the cornea and found that irradiation of the bubble-films allows spatial and selective killing of corneal cells. As i) the photosensitizer is embedded in the films, which reduces its uptake by cells and spreading into tissues and ii) the bubble-films can be removed from the tissue after laser treatment, while iii) a low laser fluence is sufficient to generate vapor bubbles, it is foreseen that bubble-films might become promising for safe resection of superficial tumors.
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http://dx.doi.org/10.1002/adma.202008379DOI Listing
May 2021

Non-viral transfection technologies for next-generation therapeutic T cell engineering.

Biotechnol Adv 2021 Jul-Aug;49:107760. Epub 2021 Apr 28.

Laboratory of General Biochemistry & Physical Pharmacy, Ghent University, Ottergemsesteenweg 460, 9000 Ghent, Belgium. Electronic address:

Genetically engineered T cells have sparked interest in advanced cancer treatment, reaching a milestone in 2017 with two FDA-approvals for CD19-directed chimeric antigen receptor (CAR) T cell therapeutics. It is becoming clear that the next generation of CAR T cell therapies will demand more complex engineering strategies and combinations thereof, including the use of revolutionary gene editing approaches. To date, manufacturing of CAR T cells mostly relies on γ-retroviral or lentiviral vectors, but their use is associated with several drawbacks, including safety issues, high manufacturing cost and vector capacity constraints. Non-viral approaches, including membrane permeabilization and carrier-based techniques, have therefore gained a lot of interest to replace viral transductions in the manufacturing of T cell therapeutics. This review provides an in-depth discussion on the avid search for alternatives to viral vectors, discusses key considerations for T cell engineering technologies, and provides an overview of the emerging spectrum of non-viral transfection technologies for T cells. Strengths and weaknesses of each technology will be discussed in relation to T cell engineering. Altogether, this work emphasizes the potential of non-viral transfection approaches to advance the next-generation of genetically engineered T cells.
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http://dx.doi.org/10.1016/j.biotechadv.2021.107760DOI Listing
April 2021

Cytosolic delivery of gadolinium via photoporation enables improved in vivo magnetic resonance imaging of cancer cells.

Biomater Sci 2021 Jun 26;9(11):4005-4018. Epub 2021 Apr 26.

Laboratory of General Biochemistry and Physical Pharmacy, Faculty of Pharmaceutical Science, Ghent University, Ottergemsesteenweg 460, 9000 Ghent, Belgium.

Longitudinal in vivo monitoring of transplanted cells is crucial to perform cancer research or to assess the treatment outcome of cell-based therapies. While several bio-imaging techniques can be used, magnetic resonance imaging (MRI) clearly stands out in terms of high spatial resolution and excellent soft-tissue contrast. However, MRI suffers from low sensitivity, requiring cells to be labeled with high concentrations of contrast agents. An interesting option is to label cells with clinically approved gadolinium chelates which generate a hyperintense MR signal. However, spontaneous uptake of the label via pinocytosis results in its endosomal sequestration, leading to quenching of the T-weighted relaxation. To avoid this quenching effect, delivery of gadolinium chelates directly into the cytosol via electroporation or hypotonic cell swelling have been proposed. However, these methods are also accompanied by several drawbacks such as a high cytotoxicity, and changes in gene expression and phenotype. Here, we demonstrate that nanoparticle-sensitized laser induced photoporation forms an attractive alternative to efficiently deliver the contrast agent gadobutrol into the cytosol of both HeLa and SK-OV-3 IP1 cells. After intracellular delivery by photoporation the quenching effect is clearly avoided, leading to a strong increase in the hyperintense T-weighted MR signal. Moreover, when compared to nucleofection as a state-of-the-art electroporation platform, photoporation has much less impact on cell viability, which is extremely important for reliable cell tracking studies. Additional experiments confirm that photoporation does not induce any change in the long-term viability or the migratory capacity of the cells. Finally, we show that gadolinium 'labeled' SK-OV-3 IP1 cells can be imaged in vivo by MRI with high soft-tissue contrast and spatial resolution, revealing indications of potential tumor invasion or angiogenesis.
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http://dx.doi.org/10.1039/d1bm00479dDOI Listing
June 2021

Nanoparticle-sensitized photoporation enables inflammasome activation studies in targeted single cells.

Nanoscale 2021 Apr 26;13(13):6592-6604. Epub 2021 Mar 26.

Laboratory of General Biochemistry and Physical Pharmacy, Faculty of Pharmaceutical Science, Ghent University, 9000 Ghent, Belgium.

Inflammasomes are multi-protein complexes that guard against cellular stress and microbial infections. Inflammasome activation studies frequently require delivery of pathogen-derived virulence factors into the cytosol of macrophages and other innate immune cells. This is a challenging requirement since primary macrophages are difficult-to-transfect, especially when it comes to the intracellular delivery of proteins. Here, we report on the use of nanoparticle-sensitized photoporation as a promising upcoming intracellular delivery technology for delivering proteins of various molecular weights into the cytosol of primary macrophages. While 60-70 nm gold nanoparticles are the most commonly used sensitizing nanoparticles for photoporation, here we find that 0.5 μm iron oxide nanoparticles perform markedly better on primary macrophages. We demonstrate that LFn-FlaA or lipopolysaccharides can be delivered in primary macrophages resulting in activation of the NLRC4 or the non-canonical inflammasome, respectively. We furthermore show that photoporation can be used for targeted delivery of these toxins into selected cells, opening up the possibility to study the interaction between inflammasome activated cells and surrounding healthy cells. Taken together, these results show that nanoparticle-sensitized photoporation is very well suited to deliver pathogenic virulence factors in primary macrophages, thus constituting an effective new enabling technology for inflammasome activation studies.
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http://dx.doi.org/10.1039/d0nr05067aDOI Listing
April 2021

The dawn of mRNA vaccines: The COVID-19 case.

J Control Release 2021 05 30;333:511-520. Epub 2021 Mar 30.

Ghent Research Group on Nanomedicines, Faculty of Pharmacy, Ghent University, Ottergemsesteenweg 460, 9000 Ghent, Belgium; Cancer Research Institute Ghent (CRIG), Ghent University, 9000 Ghent, Belgium.

In less than one year since the outbreak of the COVID-19 pandemic, two mRNA-based vaccines, BNT162b2 and mRNA-1273, were granted the first historic authorization for emergency use, while another mRNA vaccine, CVnCoV, progressed to phase 3 clinical testing. The COVID-19 mRNA vaccines represent a new class of vaccine products, which consist of synthetic mRNA strands encoding the SARS-CoV-2 Spike glycoprotein, packaged in lipid nanoparticles to deliver mRNA to cells. This review digs deeper into the scientific breakthroughs of the last decades that laid the foundations for the rapid rise of mRNA vaccines during the COVID-19 pandemic. As well as providing momentum for mRNA vaccines, SARS-CoV-2 represents an ideal case study allowing to compare design-activity differences between the different mRNA vaccine candidates. Therefore, a detailed overview of the composition and (pre)clinical performance of the three most advanced mRNA vaccines is provided and the influence of choices in their structural design on to their immunogenicity and reactogenicity profile is discussed in depth. In addition to the new fundamental insights in the mRNA vaccines' mode of action highlighted here, we also point out which unknowns remain that require further investigation and possibly, optimization in future mRNA vaccine development.
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http://dx.doi.org/10.1016/j.jconrel.2021.03.043DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8008785PMC
May 2021

Surfactant Protein B Promotes Cytosolic SiRNA Delivery by Adopting a Virus-like Mechanism of Action.

ACS Nano 2021 05 16;15(5):8095-8109. Epub 2021 Mar 16.

Ghent Research Group on Nanomedicines, Laboratory of General Biochemistry and Physical Pharmacy, Faculty of Pharmaceutical Sciences, Ghent University, Ottergemsesteenweg 460, 9000 Ghent, Belgium.

RNA therapeutics are poised to revolutionize medicine. To unlock the full potential of RNA drugs, safe and efficient (nano)formulations to deliver them inside target cells are required. Endosomal sequestration of nanocarriers represents a major bottleneck in nucleic acid delivery. Gaining more detailed information on the intracellular behavior of RNA nanocarriers is crucial to rationally develop delivery systems with improved therapeutic efficiency. Surfactant protein B (SP-B) is a key component of pulmonary surfactant (PS), essential for mammalian breathing. In contrast to the general belief that PS should be regarded as a barrier for inhaled nanomedicines, we recently discovered the ability of SP-B to promote gene silencing by siRNA-loaded and lipid-coated nanogels. However, the mechanisms governing this process are poorly understood. The major objective of this work was to obtain mechanistic insights into the SP-B-mediated cellular delivery of siRNA. To this end, we combined siRNA knockdown experiments, confocal microscopy, and focused ion beam scanning electron microscopy imaging in an non-small-cell lung carcinoma model with lipid mixing assays on vesicles that mimic the composition of (intra)cellular membranes. Our work highlights a strong correlation between SP-B-mediated fusion with anionic endosomal membranes and cytosolic siRNA delivery, a mode of action resembling that of certain viruses and virus-derived cell-penetrating peptides. Building on these gained insights, we optimized the SP-B proteolipid composition, which dramatically improved delivery efficiency. Altogether, our work provides a mechanistic understanding of SP-B-induced perturbation of intracellular membranes, offering opportunities to fuel the rational design of SP-B-inspired RNA nanoformulations for inhalation therapy.
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http://dx.doi.org/10.1021/acsnano.0c04489DOI Listing
May 2021

Delivery of Oligonucleotides into Bacteria by Fusogenic Liposomes.

Methods Mol Biol 2021 ;2246:87-96

LEPABE - Laboratory for Process Engineering, Environment, Biotechnology and Energy, Department of Chemical Engineering, Faculty of Engineering, University of Porto, Porto, Portugal.

Oligonucleotides able to hybridize bacterial RNA via in situ hybridization may potentially act as new antimicrobials, replacing antibiotics, and as fast in vivo diagnostic probes, outperforming current clinical methodologies. Nonetheless, oligonucleotides are not able to efficiently permeate the multi-layered bacterial envelope to reach their target RNA in the cytosol. Cationic fusogenic liposomes are here suggested as vehicles to enable the internalization of oligonucleotides in bacteria. Here, we describe the formulation of DOTAP-DOPE liposomes, their complexation with small negatively charged oligonucleotides, and the evaluation of the intracellular delivery of the oligonucleotides in bacteria. This strategy uncovers the potential of performing FISH in vivo for real-time detection and treatment of infections.
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http://dx.doi.org/10.1007/978-1-0716-1115-9_6DOI Listing
March 2021

Vaccinia Virus Protein B18R: Influence on mRNA Immunogenicity and Translation upon Non-Viral Delivery in Different Ocular Cell Types.

Pharmaceutics 2021 Jan 7;13(1). Epub 2021 Jan 7.

Lab of General Biochemistry and Physical Pharmacy, Faculty of Pharmaceutical Sciences, Ghent University, Ottergemsesteenweg 460, 9000 Ghent, Belgium.

In the last few years, interest has grown in the use of nucleic acids as an ocular therapy for retinal genetic diseases. Recently, our research group has demonstrated that mRNA delivery could result in effective protein expression in ocular cells following subretinal injection. Yet, although mRNA therapy comes with many advantages, its immunogenicity resulting in hampered mRNA translation delays development to the clinic. Therefore, several research groups investigate possible strategies to reduce this innate immunity. In this study, we focus on B18R, an immune inhibitor to suppress the mRNA-induced innate immune responses in two ocular cell types. We made use of retinal pigment epithelial (RPE) cells and Müller cells both as immortalized cell lines and primary bovine cells. When cells were co-incubated with both B18R and mRNA-MessengerMAX lipoplexes we observed an increase in transfection efficiency accompanied by a decrease in interferon-β production, except for the Müller cells. Moreover, uptake efficiency and cell viability were not hampered. Taken together, we showed that the effect of B18R is cell type-dependent but remains a possible strategy to improve mRNA translation in RPE cells.
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http://dx.doi.org/10.3390/pharmaceutics13010074DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7827308PMC
January 2021

Long-term live-cell microscopy with labeled nanobodies delivered by laser-induced photoporation.

Nano Res 2020 Feb 18;13(2):485-495. Epub 2020 Jan 18.

Laboratory of General Biochemistry and Physical Pharmacy, Faculty of Pharmacy, Ghent University, Ghent B-9000, Belgium.

Fluorescence microscopy is the method of choice for studying intracellular dynamics. However, its success depends on the availability of specific and stable markers. A prominent example of markers that are rapidly gaining interest are nanobodies (Nbs, ~ 15 kDa), which can be functionalized with bright and photostable organic fluorophores. Due to their relatively small size and high specificity, Nbs offer great potential for high-quality long-term subcellular imaging, but suffer from the fact that they cannot spontaneously cross the plasma membrane of live cells. We have recently discovered that laser-induced photoporation is well suited to deliver extrinsic labels to living cells without compromising their viability. Being a laser-based technology, it is readily compatible with light microscopy and the typical cell recipients used for that. Spurred by these promising initial results, we demonstrate here for the first time successful long-term imaging of specific subcellular structures with labeled nanobodies in living cells. We illustrate this using Nbs that target GFP/YFP-protein constructs accessible in the cytoplasm, actin-bundling protein Fascin, and the histone H2A/H2B heterodimers. With an efficiency of more than 80% labeled cells and minimal toxicity (~ 2%), photoporation proved to be an excellent intracellular delivery method for Nbs. Time-lapse microscopy revealed that cell division rate and migration remained unaffected, confirming excellent cell viability and functionality. We conclude that laser-induced photoporation labeled Nbs can be easily delivered into living cells, laying the foundation for further development of a broad range of Nbs with intracellular targets as a toolbox for long-term live-cell microscopy.
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http://dx.doi.org/10.1007/s12274-020-2633-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7116313PMC
February 2020

Ocular barriers to retinal delivery of intravitreal liposomes: Impact of vitreoretinal interface.

J Control Release 2020 12 19;328:952-961. Epub 2020 Oct 19.

Drug Research Programme, Division of Pharmaceutical Biosciences, Faculty of Pharmacy, University of Helsinki, Helsinki, Finland; School of Pharmacy, Faculty of Health Sciences, University of Eastern Finland, Kuopio, Finland; Institute of Chemistry, St Petersburg State University, Petergoff, St Petersburg, Russian Federation.

Drug delivery to the posterior segment of the eye is challenging due to several anatomical and physiological barriers. Thus, there is a need for prolonged action and targeted drug delivery to treat retinal diseases. Intravitreal injections avoid anterior eye barriers, but the vitreoretinal interface and inner limiting membrane (ILM) may prevent access of drug delivery systems to the retina. Existing data on retinal permeation of intravitreal nanoparticles are sparse and probably misleading due to the inter-species differences of retinal structures in rodents and humans. To bridge this gap, retinal permeation of light-activated liposomes was studied in an ex vivo bovine explant system that simulates the structure of vitreoretinal interface and intact ILM. Our findings indicate that the particle size plays a significant role in determining the retinal penetration as the liposomes of >100 nm sized failed to overcome the ILM and could not permeate into the retina. In addition, our results demonstrate the impact of surface charge and PEG-coating on retinal penetration. Small (≈ 50 nm) anionic liposomes with PEG coating showed the most extensive distribution and cellular localization in the retina. In summary, this study extends understanding of ocular barriers, and provides valuable information to augment design of retinal drug delivery systems.
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http://dx.doi.org/10.1016/j.jconrel.2020.10.028DOI Listing
December 2020

Lyophilization and nebulization of pulmonary surfactant-coated nanogels for siRNA inhalation therapy.

Eur J Pharm Biopharm 2020 Dec 3;157:191-199. Epub 2020 Oct 3.

Ghent Research Group on Nanomedicines, Laboratory of General Biochemistry and Physical Pharmacy, Faculty of Pharmaceutical Sciences, Department of Pharmaceutics, Ghent University, Ottergemsesteenweg 460, 9000 Ghent, Belgium. Electronic address:

RNA interference (RNAi) enables highly specific silencing of potential target genes for treatment of pulmonary pathologies. The intracellular RNAi pathway can be activated by cytosolic delivery of small interfering RNA (siRNA), inducing sequence-specific gene knockdown on the post-transcriptional level. Although siRNA drugs hold many advantages over currently applied therapies, their clinical translation is hampered by inefficient delivery across cellular membranes. We previously developed hybrid nanoparticles consisting of an siRNA-loaded nanosized hydrogel core (nanogel) coated with Curosurf®, a clinically used pulmonary surfactant (PS). The latter enhances both particle stability as well as intracellular siRNA delivery, which was shown to be governed by the PS-associated surfactant protein B (SP-B). Despite having a proven in vitro and in vivo siRNA delivery potential when prepared ex novo, clinical translation of this liquid nanoparticle suspension requires the identification of a long-term preservation strategy that maintains nanoparticle stability and potency. In addition, to achieve optimal pulmonary deposition of the nanocomposite, its compatibility with state-of-the-art pulmonary administration techniques should be evaluated. Here, we demonstrate that PS-coated nanogels can be lyophilized, reconstituted and subsequently nebulized via a vibrating mesh nebulizer. The particles retain their physicochemical integrity and their ability to deliver siRNA in a human lung epithelial cell line. The latter result suggests that the functional integrity of SP-B in the PS coat towards siRNA delivery might be preserved as well. Of note, successful lyophilization was achieved without the need for stabilizing lyo- or cryoprotectants. Our results demonstrate that PS-coated siRNA-loaded nanogels can be lyophilized, which offers the prospect of long-term storage. In addition, the formulation was demonstrated to be suitable for local administration with a state-of-the-art nebulizer for human use upon reconstitution. Hence, the data presented in this study represent an important step towards clinical application of such nanocomposites for treatment of pulmonary disease.
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http://dx.doi.org/10.1016/j.ejpb.2020.09.011DOI Listing
December 2020

Physical transfection technologies for macrophages and dendritic cells in immunotherapy.

Expert Opin Drug Deliv 2021 02 19;18(2):229-247. Epub 2020 Oct 19.

Laboratory of General Biochemistry and Physical Pharmacy, Ghent University, Ghent, Belgium.

Introduction: Dendritic cells (DCs) and macrophages, two important antigen presenting cells (APCs) of the innate immune system, are being explored for the use in cell-based cancer immunotherapy. For this application, the therapeutic potential of patient-derived APCs is increased by delivering different types of functional macromolecules, such as mRNA and pDNA, into their cytosol. Compared to the use of viral and non-viral delivery vectors, physical intracellular delivery techniques are known to be more straightforward, more controllable, faster and generate high delivery efficiencies.

Areas Covered: This review starts with electroporation as the most traditional physical transfection method, before continuing with the more recent technologies such as sonoporation, nanowires and microfluidic cell squeezing. A description is provided of each of those intracellular delivery technologies with their strengths and weaknesses, especially paying attention to delivery efficiency and safety profile.

Expert Opinion: Given the common use of electroporation for the production of therapeutic APCs, it is recommended that more detailed studies are performed on the effect of electroporation on APC fitness, even down to the genetic level. Newer intracellular delivery technologies seem to have less impact on APC functionality but further work is needed to fully uncover their suitability to transfect APCs with different types of macromolecules.
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http://dx.doi.org/10.1080/17425247.2021.1828340DOI Listing
February 2021

Synergy between Intraperitoneal Aerosolization (PIPAC) and Cancer Nanomedicine: Cisplatin-Loaded Polyarginine-Hyaluronic Acid Nanocarriers Efficiently Eradicate Peritoneal Metastasis of Advanced Human Ovarian Cancer.

ACS Appl Mater Interfaces 2020 Jul 22;12(26):29024-29036. Epub 2020 Jun 22.

Laboratory of General Biochemistry and Physical Pharmacy, Faculty of Pharmaceutical Sciences, Ghent University, 9000 Ghent, Belgium.

Intra-abdominal dissemination of peritoneal nodules, a condition known as peritoneal carcinomatosis (PC), is typically diagnosed in ovarian cancer patients at the advanced stages. The current treatment of PC consists of perioperative systemic chemotherapy and cytoreductive surgery, followed by intra-abdominal flushing with solutions of chemotherapeutics such as cisplatin and oxaliplatin. In this study, we developed cisplatin-loaded polyarginine-hyaluronic acid nanoscale particles (Cis-pARG-HA NPs) with high colloidal stability, marked drug loading efficiency, unimpaired biological activity, and tumor-targeting ability. Injected Cis-pARG-HA NPs showed enhanced antitumor activity in a rat model of PC, compared to injection of the free cisplatin drug. The activity of Cis-pARG-HA NPs could even be further improved when administered by an intra-abdominal aerosol therapy, referred to as pressurized intraperitoneal aerosol chemotherapy (PIPAC). PIPAC is hypothesized to ensure a more homogeneous drug distribution together with a deeper drug penetration into peritoneal tumor nodules within the abdominal cavity. Using fluorescent pARG-HA NPs, this enhanced nanoparticle deposit on tumors could indeed be observed in regions opposite the aerosolization nozzle. Therefore, this study demonstrates that nanoparticles carrying chemotherapeutics can be synergistically combined with the PIPAC technique for IP therapy of disseminated advanced ovarian tumors, while this synergistic effect was not observed for the administration of free cisplatin.
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http://dx.doi.org/10.1021/acsami.0c05554DOI Listing
July 2020

Fluorescence-Based Quantification of Messenger RNA and Plasmid DNA Decay Kinetics in Extracellular Biological Fluids and Cell Extracts.

Adv Biosyst 2020 05 23;4(5):e2000057. Epub 2020 Mar 23.

Laboratory of General Biochemistry and Physical Pharmacy, Faculty of Pharmaceutical Sciences, Ghent University, Ghent, 9000, Belgium.

Extracellular and intracellular degradation of nucleic acids remains an issue in non-viral gene therapy. Understanding biodegradation is critical for the rational design of gene therapeutics in order to maintain stability and functionality at the target site. However, there are only limited methods available that allow determining the stability of genetic materials in biological environments. In this context, the decay kinetics of fluorescently labeled plasmid DNA (pDNA) and messenger RNA (mRNA) in undiluted biological samples (i.e., human serum, human ascites, bovine vitreous) and cell extracts is studied using fluorescence correlation spectroscopy (FCS) and single particle tracking (SPT). It is demonstrated that FCS is suitable to follow mRNA degradation, while SPT is better suited to investigate pDNA integrity. The half-life of mRNA and pDNA is ≈1-2 min and 1-4 h in biological samples, respectively. The resistance against biodegradation drastically improves by complexation with lipid-based carriers. Taken together, FCS and SPT are able to quantify the integrity of mRNA and pDNA, respectively, as a function of time, both in the extracellular biological fluids and cell extracts. This in turn allows to focus on the important but less understood issue of nucleic acids degradation in more detail and to rationally optimize gene delivery system as therapeutics.
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http://dx.doi.org/10.1002/adbi.202000057DOI Listing
May 2020

Faithful Fabrication of Biocompatible Multicompartmental Memomicrospheres for Digitally Color-Tunable Barcoding.

Small 2020 06 10;16(24):e1907586. Epub 2020 May 10.

Joint Laboratory of Advanced Biomedical Technology (NFU-UGent), College of Chemical Engineering, Nanjing Forestry University (NFU), Nanjing, 210037, P. R. China.

Barcodes have attracted widespread attention, especially for the multiplexed bioassays and anti-counterfeiting used toward medical and biomedical applications. An enabling gas-shearing approach is presented for generating 10-faced microspherical barcodes with precise control over the properties of each compartment. As such, the color of each compartment could be programmatically adjusted in the 10-faced memomicrospheres by using pregel solutions containing different combinations of fluorescent nanoparticles. During the process, three primary colors (red, green, and blue) are adopted to obtain up to seven merged fluorescent colors for constituting a large amount of coding as well as a magnetic compartment, capable of effective and robust high-throughput information-storage. More importantly, by using the biocompatible sodium alginate to construct the multicolor microspherical barcodes, the proposed technology is likely to advance the fields of food and pharmaceutics anti-counterfeiting. These remarkable properties point to the potential value of gas-shearing in engineering microspherical barcodes for biomedical applications in the future.
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http://dx.doi.org/10.1002/smll.201907586DOI Listing
June 2020

Intracellular Labeling with Extrinsic Probes: Delivery Strategies and Applications.

Small 2020 06 30;16(22):e2000146. Epub 2020 Apr 30.

Laboratory of General Biochemistry and Physical Pharmacy, Faculty of Pharmacy, Ghent University, Ghent, B-9000, Belgium.

Extrinsic probes have outstanding properties for intracellular labeling to visualize dynamic processes in and of living cells, both in vitro and in vivo. Since extrinsic probes are in many cases cell-impermeable, different biochemical, and physical approaches have been used to break the cell membrane barrier for direct delivery into the cytoplasm. In this Review, these intracellular delivery strategies are discussed, briefly explaining the mechanisms and how they are used for live-cell labeling applications. Methods that are discussed include three biochemical agents that are used for this purpose-purpose-different nanocarriers, cell penetrating peptides and the pore-foraming bacterial toxin streptolysin O. Most successful intracellular label delivery methods are, however, based on physical principles to permeabilize the membrane and include electroporation, laser-induced photoporation, micro- and nanoinjection, nanoneedles or nanostraws, microfluidics, and nanomachines. The strengths and weaknesses of each strategy are discussed with a systematic comparison provided. Finally, the extrinsic probes that are reported for intracellular labeling so-far are summarized, together with the delivery strategies that are used and their performance. This combined information should provide for a useful guide for choosing the most suitable delivery method for the desired probes.
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http://dx.doi.org/10.1002/smll.202000146DOI Listing
June 2020

Cationic Amphiphilic Drugs Boost the Lysosomal Escape of Small Nucleic Acid Therapeutics in a Nanocarrier-Dependent Manner.

ACS Nano 2020 04 14;14(4):4774-4791. Epub 2020 Apr 14.

Ghent Research Group on Nanomedicines, Laboratory of General Biochemistry and Physical Pharmacy, Faculty of Pharmaceutical Sciences, Ghent University, Ottergemsesteenweg 460, 9000 Ghent, Belgium.

Small nucleic acid (NA) therapeutics, such as small interfering RNA (siRNA), are generally formulated in nanoparticles (NPs) to overcome the multiple extra- and intracellular barriers upon administration. Interaction with target cells typically triggers endocytosis and sequesters the NPs in endosomes, thus hampering the pharmacological activity of the encapsulated siRNAs that occurs in the cytosol. Unfortunately, for most state-of-the-art NPs, endosomal escape is largely inefficient. As a result, the bulk of the endocytosed NA drug is rapidly trafficked toward the degradative lysosomes that are considered as a dead end for siRNA nanomedicines. In contrast to this paradigm, we recently reported that cationic amphiphilic drugs (CADs) could strongly promote functional siRNA delivery from the endolysosomal compartment transient induction of lysosomal membrane permeabilization. However, many questions still remain regarding the broader applicability of such a CAD adjuvant effect on NA delivery. Here, we report a drug repurposing screen (National Institutes of Health Clinical Collection) that allowed identification of 56 CAD adjuvants. We furthermore demonstrate that the CAD adjuvant effect is dependent on the type of nanocarrier, with NPs that generate an appropriate pool of decomplexed siRNA in the endolysosomal compartment being most susceptible to CAD-promoted gene silencing. Finally, the CAD adjuvant effect was verified on human ovarian cancer cells and for antisense oligonucleotides. In conclusion, this study strongly expands our current knowledge on how CADs increase the cytosolic release of small NAs, providing relevant insights to more rationally combine CAD adjuvants with NA-loaded NPs for future therapeutic applications.
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http://dx.doi.org/10.1021/acsnano.0c00666DOI Listing
April 2020

Materials and Technologies to Combat Counterfeiting of Pharmaceuticals: Current and Future Problem Tackling.

Adv Mater 2020 Mar 3;32(11):e1905486. Epub 2020 Feb 3.

Joint Laboratory of Advanced Biomedical Technology (NFU-UGent), College of Chemical Engineering, Nanjing Forestry University, 210037, Nanjing, P. R. China.

The globalization of drug trade leads to the expansion of pharmaceutical counterfeiting. The immense threat of low quality drugs to millions of patients is considered to be an under-addressed global health challenge. Analytical authentication technologies are the most effective methods to identify active pharmaceutical ingredients and impurities. However, most of these analytical testing techniques are expensive and need skilled personnel. To combat counterfeiting of drugs, the package of an increasing number of drugs is being protected through advanced package labeling technologies. Though, package labeling is only effective if the drugs are not repackaged. Therefore "in-drug labeling," instead of "drug package labeling," may become powerful tools to protect drugs. This review aims to overview how advanced micro- and nanomaterials might become interesting markers for the labeling of tablets and capsules. Clearly, how well such identifiers can be integrated into "solid drugs" without compromising drug safety and efficacy remains a challenge. Also, incorporation of tags has so far only been reported for the protection of solid drug dosage forms. No doubts that in-drug labeling technologies for "liquid drugs," like injectables which contain expensive peptides, monoclonal antibodies, vaccines, dermal fillers, could help to protect them from counterfeiting as well.
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http://dx.doi.org/10.1002/adma.201905486DOI Listing
March 2020

Merging the best of two worlds: New excipients and old sugars deliver mRNA into lungs.

J Control Release 2019 11 1;314:177-178. Epub 2019 Nov 1.

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http://dx.doi.org/10.1016/j.jconrel.2019.10.055DOI Listing
November 2019

Longitudinal In Vivo Assessment of Host-Microbe Interactions in a Murine Model of Pulmonary Aspergillosis.

iScience 2019 Oct 18;20:184-194. Epub 2019 Sep 18.

Biomedical MRI/Molecular Small Animal Imaging Center (MoSAIC), KU Leuven, Leuven, Belgium. Electronic address:

The fungus Aspergillus fumigatus is ubiquitous in nature and the most common cause of invasive pulmonary aspergillosis (IPA) in patients with a compromised immune system. The development of IPA in patients under immunosuppressive treatment or in patients with primary immunodeficiency demonstrates the importance of the host immune response in controlling aspergillosis. However, study of the host-microbe interaction has been hampered by the lack of tools for their non-invasive assessment. We developed a methodology to study the response of the host's immune system against IPA longitudinally in vivo by using fluorine-19 magnetic resonance imaging (F MRI). We showed the advantage of a perfluorocarbon-based contrast agent for the in vivo labeling of macrophages and dendritic cells, permitting quantification of pulmonary inflammation in different murine IPA models. Our findings reveal the potential of F MRI for the assessment of rapid kinetics of innate immune response against IPA and the permissive niche generated through immunosuppression.
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http://dx.doi.org/10.1016/j.isci.2019.09.022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6817634PMC
October 2019

Lifting shadows off intracellular processing.

J Control Release 2019 09 9;310:209-210. Epub 2019 Sep 9.

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http://dx.doi.org/10.1016/j.jconrel.2019.09.003DOI Listing
September 2019

Nanocarrier Lipid Composition Modulates the Impact of Pulmonary Surfactant Protein B (SP-B) on Cellular Delivery of siRNA.

Pharmaceutics 2019 Aug 23;11(9). Epub 2019 Aug 23.

Ghent Research Group on Nanomedicines, Laboratory of General Biochemistry and Physical Pharmacy, Department of Pharmaceutics, Ghent University, Ottergemsesteenweg 460, 9000 Ghent, Belgium.

Two decades since the discovery of the RNA interference (RNAi) pathway, we are now witnessing the approval of the first RNAi-based treatments with small interfering RNA (siRNA) drugs. Nevertheless, the widespread use of siRNA is limited by various extra- and intracellular barriers, requiring its encapsulation in a suitable (nanosized) delivery system. On the intracellular level, the endosomal membrane is a major barrier following endocytosis of siRNA-loaded nanoparticles in target cells and innovative materials to promote cytosolic siRNA delivery are highly sought after. We previously identified the endogenous lung surfactant protein B (SP-B) as siRNA delivery enhancer when reconstituted in (proteo) lipid-coated nanogels. It is known that the surface-active function of SP-B in the lung is influenced by the lipid composition of the lung surfactant. Here, we investigated the role of the lipid component on the siRNA delivery-promoting activity of SP-B proteolipid-coated nanogels in more detail. Our results clearly indicate that SP-B prefers fluid membranes with cholesterol not exceeding physiological levels. In addition, SP-B retains its activity in the presence of different classes of anionic lipids. In contrast, comparable fractions of SP-B did not promote the siRNA delivery potential of DOTAP:DOPE cationic liposomes. Finally, we demonstrate that the beneficial effect of lung surfactant on siRNA delivery is not limited to lung-related cell types, providing broader therapeutic opportunities in other tissues as well.
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http://dx.doi.org/10.3390/pharmaceutics11090431DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6781292PMC
August 2019

Sonoprinting of nanoparticle-loaded microbubbles: Unraveling the multi-timescale mechanism.

Biomaterials 2019 10 27;217:119250. Epub 2019 Jun 27.

Laboratory of General Biochemistry and Physical Pharmacy, Ghent Research Group on Nanomedicine, Faculty of Pharmaceutical Sciences, Ghent University, Ottergemsesteenweg 460, Ghent, Belgium.

Ultrasound-triggered microbubble-assisted drug delivery is a promising tool for localized therapy. Several studies have shown the potential of nanoparticle-loaded microbubbles to effectively enhance the delivery of therapeutic agents to target tissue. We recently discovered that nanoparticle-carrying microbubbles can deposit the nanoparticles in patches onto cell membranes, a process which we termed 'sonoprinting'. However, the biophysical mechanisms behind sonoprinting are not entirely clear. In addition, the question remains how the ultrasound parameters, such as acoustic pressure and pulse duration, influence sonoprinting. Aiming for a better understanding of sonoprinting, this report investigates the behavior of nanoparticle-loaded microbubbles under ultrasound exposure, making use of three advanced optical imaging techniques with frame rates ranging from 5 frames per second to 10 million frames per second, to capture the biophysical cell-bubble interactions that occur on a multitude of timescales. We observed that non-spherically oscillating microbubbles release their nanoparticle payload in the first few cycles of ultrasound insonation. At low acoustic pressures, the released nanoparticles are transported away from the cells by microstreaming, which does not favor uptake of the nanoparticles by the cells. However, higher acoustic pressures (>300 kPa) and longer ultrasound pulses (>100 cycles) lead to rapid translation of the microbubbles, due to acoustic radiation forces. As a result, the released nanoparticles are transported along in the wake of the microbubbles, which eventually leads to the deposition of nanoparticles in elongated patches on the cell membrane, i.e. sonoprinting. We conclude that a sufficiently high acoustic pressure and long pulses are needed for sonoprinting of nanoparticles on cells.
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http://dx.doi.org/10.1016/j.biomaterials.2019.119250DOI Listing
October 2019

Photoablation of Human Vitreous Opacities by Light-Induced Vapor Nanobubbles.

ACS Nano 2019 07 12;13(7):8401-8416. Epub 2019 Jul 12.

Laboratory of General Biochemistry & Physical Pharmacy, Faculty of Pharmaceutical Sciences , Ghent University , Ottergemsesteenweg 460 , Ghent 9000 , Belgium.

Myopia, diabetes, and aging are the main causes of progressive vitreous collagen aggregation, resulting in vitreous opacities, which can significantly disturb vision. As vitreous opacities, which induce the visual phenomenon of "floaters", are accessible with nanomaterials and light, we propose a nanotechnology-based approach to locally ablate them with highly reduced light energy compared to the more traditional YAG laser therapy. Our strategy relies on the plasmon properties of gold nanoparticles that generate vapor nanobubbles upon pulsed-laser illumination whose mechanical force can ablate vitreous opacities. We designed gold nanoparticles coated with hyaluronic acid (HA), which have excellent diffusional mobility in human vitreous, an essential requirement to reach the vitreous opacities. In addition, we found that HA-coated gold nanoparticles can accumulate extensively on human vitreous opacities that were obtained by vitrectomy from patients with vision-degrading myodesopsia. When subsequently applying nanosecond laser pulses, the collagen aggregates were efficiently destroyed with ∼1000 times less light energy than typically used in YAG laser therapy. This low-energy "floater-specific destruction", which is due to the accumulation of the small gold nanoparticles on the opacities, is attractive, as it may be safer to the surrounding ocular tissues while at the same time being easier and faster to apply compared to YAG laser therapy, where the opacities need to be ablated piece by piece by a tightly focused laser beam. Gold nanoparticle-assisted photoablation may therefore provide a safer, faster, and more reliable destruction of vitreous opacities in the treatment of ophthalmologic diseases.
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http://dx.doi.org/10.1021/acsnano.9b04050DOI Listing
July 2019

Non-viral delivery of chemically modified mRNA to the retina: Subretinal versus intravitreal administration.

J Control Release 2019 08 29;307:315-330. Epub 2019 Jun 29.

Ghent Research Group on Nanomedicines, Laboratory of General Biochemistry and Physical Pharmacy, Ghent University, Ottergemsesteenweg 460, 9000 Ghent, Belgium; Cancer Research Institute Ghent (CRIG), Ghent University Hospital, De Pintelaan 185, 9000 Ghent, Belgium. Electronic address:

mRNA therapeutics have recently experienced a new wave of interest, mainly due to the discovery that chemical modifications to mRNA's molecular structure could drastically reduce its inherent immunogenicity and perceived instability. On this basis, we aimed to explore the potential of chemically stabilized mRNA for ocular applications. More specifically, we investigated the behavior of mRNA-loaded lipid-based carriers in human retinal cells (in vitro), in bovine retinal explants (ex vivo) and in mouse retinas (in vivo). We demonstrate a clear superiority of mRNA over pDNA to induce protein expression in different retinal cell types, which was further enhanced by chemical modification of the mRNA, providing up to ~1800-fold higher reporter gene expression compared to pDNA. Moreover, transgene expression could be detected for at least 20 days after a single administration of chemically modified mRNA in vitro. We furthermore determined the localization and extent of mRNA expression depending on the administration route. After subretinal (SR) administration, mRNA expression was observed in vivo and ex vivo. By contrast, intravitreal (IVT) administration resulted in limited expression in vivo. Using ex vivo bovine explants with an intact vitreoretinal (VR) interface we could attribute this to the inner limiting membrane (ILM), which presents a large barrier for non-viral delivery of mRNA, trapping mRNA complexes at the vitreal side. When the vitreous was removed, which compromises the ILM, mRNA expression was apparent and seemed to colocalize with Müller cells or photoreceptors after respectively IVT or SR administration. Taken together, this study represents a first step towards mRNA-mediated therapy for retinal diseases.
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http://dx.doi.org/10.1016/j.jconrel.2019.06.042DOI Listing
August 2019

Aerosolization of Nanotherapeutics as a Newly Emerging Treatment Regimen for Peritoneal Carcinomatosis.

Cancers (Basel) 2019 Jun 28;11(7). Epub 2019 Jun 28.

Laboratory of General Biochemistry and Physical Pharmacy, Faculty of Pharmaceutical Sciences, Ghent University, 9000 Ghent, Belgium.

Recent advances in locoregional chemotherapy have opened the door to new approaches for the clinical management of peritoneal carcinomatosis (PC) by facilitating the delivery of anti-neoplastic agents directly to the tumor site, while mitigating adverse effects typically associated with systemic administration. In particular, an innovative intra-abdominal chemotherapeutic approach, known as Pressurized Intraperitoneal Aerosol Chemotherapy (PIPAC), was recently introduced to the intraperitoneal (IP) therapy regimens as a palliative therapeutic option in patients with PC, presumably providing a better drug distribution pattern together with deeper drug penetration into tumor nodules within the peritoneal space. Furthermore, the progress of nanotechnology in the past few decades has prompted the application of different nanomaterials in IP cancer therapy, offering new possibilities in this field ranging from an extended retention time to sustained drug release in the peritoneal cavity. This review highlights the progress, challenges, and opportunities in utilizing cancer nanotherapeutics for locoregional drug delivery, with a special emphasis on the aerosolization approach for intraperitoneal therapies.
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http://dx.doi.org/10.3390/cancers11070906DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6678324PMC
June 2019

High Pressure Nebulization (PIPAC) Versus Injection for the Intraperitoneal Administration of mRNA Complexes.

Pharm Res 2019 Jun 24;36(9):126. Epub 2019 Jun 24.

Laboratory of General Biochemistry and Physical Pharmacy, Faculty of Pharmaceutical Sciences, Ghent University, Ottergemsesteenweg 460, 9000, Ghent, Belgium.

Purpose: Pressurized intraperitoneal aerosol chemotherapy (PIPAC) is a novel technique delivering drugs into the abdominal cavity as an aerosol under high pressure. It is hypothesized to have advantages such as enhancing tissue uptake, distributing drugs homogeneously within the closed and expanded abdominal cavity and higher local concentration of drugs in the peritoneal cavity. However, the clinical trials of PIPAC so far are limited to liquid chemotherapeutic solution, and the applicability of biomolecules (such as mRNA, siRNA and oligonucleotide) is not known. We aimed to investigate the feasibility of administrating mRNA lipoplexes to the peritoneal cavity via high pressure nebulization.

Methods: We firstly investigated the influences of nebulization on physicochemical properties and in vitro transfection efficiency of mRNA lipoplexes. Then, mRNA lipoplexes were delivered to healthy rats through intravenous injection, intraperitoneal injection and PIPAC, respectively.

Results: mRNA lipoplexes can withstand the high pressure applied during the PIPAC procedure in vitro. Bioluminescence localized to the peritoneal cavity of rats after administration by IP injection and nebulization, while intravenous injection mainly induced protein expression in the spleen.

Conclusion: This study demonstrated that local nebulization is feasible to apply mRNA complexes in the peritoneal cavity during a PIPAC procedure.
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http://dx.doi.org/10.1007/s11095-019-2646-zDOI Listing
June 2019

The obstacle course to the inner retina: Hyaluronic acid-coated lipoplexes cross the vitreous but fail to overcome the inner limiting membrane.

Eur J Pharm Biopharm 2019 Aug 28;141:161-171. Epub 2019 May 28.

Ghent Research Group on Nanomedicines, Laboratory of General Biochemistry and Physical Pharmacy, Ghent University, Ottergemsesteenweg 460, 9000 Ghent, Belgium; Cancer Research Institute Ghent (CRIG), Ghent University Hospital, De Pintelaan 185, 9000 Ghent, Belgium. Electronic address:

Considerable research over the last few years has revealed dysregulation of growth factors in various retinal diseases, such as glaucoma, diabetic retinopathy and photoreceptor degenerations. The use of messengerRNA (mRNA) to transiently overexpress a specific factor could compensate for this imbalance. However, a critical challenge of this approach lies in the ability to efficiently deliver mRNA molecules to the retinal target cells. In this study we found that intravitreal (IVT) injection is an attractive approach to deliver mRNA to the retina, providing two critical barriers can be overcome: the vitreous and the inner limiting membrane (ILM). We demonstrated that the vitreous is indeed a major hurdle in the delivery of the cationic mRNA-complexes to retinal cells, both in terms of vitreal mobility and cellular uptake. To improve their intravitreal mobility and avoid unwanted extracellular interactions, we evaluated the use of hyaluronic acid (HA) as an electrostatic coating strategy. This HA-coating provided the complexes with a negative surface charge, markedly enhancing their mobility in the vitreous humor, without reducing their cellular internalization and transfection efficiency. However, although this coating strategy allows the mRNA-complexes to successfully overcome the vitreal barrier, the majority of the particles accumulated at the ILM. This study therefore underscores the crucial barrier function of the ILM toward non-viral retinal gene delivery and the need to smartly design mRNA-carriers able to surmount the vitreous as well as the ILM.
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http://dx.doi.org/10.1016/j.ejpb.2019.05.023DOI Listing
August 2019

Gas-Shearing Fabrication of Multicompartmental Microspheres: A One-Step and Oil-Free Approach.

Adv Sci (Weinh) 2019 May 28;6(9):1802342. Epub 2019 Feb 28.

College of Chemical Engineering Jiangsu Key Lab of Biomass-based Green Fuels and Chemicals Nanjing Forestry University (NFU) Nanjing 210037 P. R. China.

Multicompartmental microparticles (MCMs) have attracted considerable attention in biomedical engineering and materials sciences, as they can carry multiple materials in the separated phases of a single particle. However, the robust fabrication of monodisperse, highly compartmental MCMs at the micro- and nanoscales remains challenging. Here, a simple one-step and oil-free process, based on the gas-flow-assisted formation of microdroplets ("gas-shearing"), is established for the scalable production of monodisperse MCMs. By changing the configuration of the needle system and gas flow in the spray ejector device, the oil-free gas-shearing process easily allows the design of microparticles consisting of two, four, six, and even eight compartments with a precise control over the properties of each compartment. As oils and surfactants are not used, the gas-shearing method is highly cytocompatible. The versatile applications of such MCMs are demonstrated by producing a magnetic microrobot and a biocompatible carrier for the coculturing of cells. This research suggests that the oil-free gas-shearing strategy is a reliable, scalable, and biofriendly process for producing MCMs that may become attractive materials for biomedical applications.
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http://dx.doi.org/10.1002/advs.201802342DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6498303PMC
May 2019