Single-step batch fabrication of microfluidic paper-based analytical devices with a 3D printer and their applications in nanoenzyme-enhanced visual detection of dopamine.
Analytical and bioanalytical chemistry
Wax printing is the most widely used method for fabricating microfluidic paper-based analytical devices (μPADs), but it still suffers from disadvantages like discontinuation of wax printers and need for additional equipment for heating treatment. To address these issues, this work initially describes a new class of wax printing approach for high-precision, batch fabrication of μPADs using a household 3D printer. It only involves a one patterning step of printing polyethylene wax into rice paper body. Under optimized parameters, a fabrication resolution, namely the minimum hydrophilic channel width, down to ~189 ± 30 μm could be achieved. In addition, the analytical applicability of such polyethylene wax-patterned μPADs was demonstrated well with enhanced colorimetric detection of dopamine as a model analyte by combining metal-organic framework (MOF) based nanoenzymes (ZIF-67) with a smartphone (for portable quantitative readout). The developed nanosensor could linearly detect dopamine over a concentration range from 10 to 1000 μM, with a detection limit of ca. 2.75 μM (3σ). The recovery results for analyzing several real samples (i.e., pig feed, chicken feed, pork and human serum) were between 91.82 and 102.79%, further validating its good detection accuracy for potential practical applications in food safety and medical diagnosis.
10.1007/s00216-024-05337-2
Lipid Nanoparticle-Based Inhibitors for SARS-CoV-2 Host Cell Infection.
International journal of nanomedicine
Purpose:The global pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the lingering threat to public health has fueled the search for effective therapeutics to treat SARS-CoV-2. This study aimed to develop lipid nanoparticle (LNP) inhibitors of SARS-CoV-2 entry to reduce viral infection in the nose and upper airway. Methods:Two types of LNP formulations were prepared following a microfluidic mixing method. The LNP-Trap consisted of DOPC, DSPC, cholesterol, and DSPE-PEG-COOH modified with various spike protein binding ligands, including ACE2 peptide, recombinant human ACE2 (rhACE2) or monoclonal antibody to spike protein (mAb). The LNP-Trim consisted of ionizing cationic DLin-MC3-DMA, DSPC, cholesterol, and DMG-PEG lipids encapsulating si or si. Both formulations were assayed for biocompatibility and cell uptake in airway epithelial cells (Calu-3). Functional assessment of activity was performed using SARS-CoV-2 spike protein binding assays (LNP-Trap), host receptor knockdown (LNP-Trim), and SARS-CoV-2 pseudovirus neutralization assay (LNP-Trap and LNP-Trim). Localization and tissue distribution of fluorescently labeled LNP formulations were assessed in mice following intranasal administration. Results:Both LNP formulations were biocompatible based on cell impedance and MTT cytotoxicity studies in Calu-3 cells at concentrations as high as 1 mg/mL. LNP-Trap formulations were able to bind spike protein and inhibit pseudovirus infection by 90% in Calu-3 cells. LNP-Trim formulations reduced ACE2 and TMPRSS2 at the mRNA (70% reduction) and protein level (50% reduction). The suppression of host targets in Calu-3 cells treated with LNP-Trim resulted in over 90% inhibition of pseudovirus infection. In vivo studies demonstrated substantial retention of LNP-Trap and LNP-Trim in the nasal cavity following nasal administration with minimal systemic exposure. Conclusion:Both LNP-Trap and LNP-Trim formulations were able to safely and effectively inhibit SARS-CoV-2 pseudoviral infection in airway epithelial cells. These studies provide proof-of-principle for a localized treatment approach for SARS-CoV-2 in the upper airway.
10.2147/IJN.S448005
Microfluidic encapsulation of enzymes and steroids within solid lipid nanoparticles.
Drug delivery and translational research
The production of solid lipid nanoparticles (SLNs) is challenging, especially when considering the incorporation of biologics. A novel in-house method of microfluidic production of biologic-encapsulated SLNs is proposed, using a variety of base materials for formulation to help overcome the barriers presented during manufacture and administration. Trypsin is used as a model drug for hydrophilic encapsulation whilst testosterone is employed as a positive non-biologic lipophilic control active pharmaceutical ingredient. Particle sizes obtained ranged from 160 to 320 nm, and a lead formulation has been identified from the combinations assayed, allowing for high encapsulation efficiencies (47-90%, respectively) of both the large hydrophilic and the small hydrophobic active pharmaceutical ingredients (APIs). Drug release profiles were analysed in vitro to provide useful insight into sustained kinetics, providing data towards future in vivo studies, which displayed a slow prolonged release for testosterone and a quicker burst release for trypsin. The study represents a large leap forward in the field of SLN production, especially in the field of difficult-to-encapsulate molecules, and the technique also benefits from being more environmentally sustainable due to the use of microfluidics.
10.1007/s13346-023-01398-5
Lipid nanoparticles loaded with ribonucleoprotein-oligonucleotide complexes synthesized using a microfluidic device exhibit robust genome editing and hepatitis B virus inhibition.
Suzuki Yuichi,Onuma Haruno,Sato Risa,Sato Yusuke,Hashiba Akari,Maeki Masatoshi,Tokeshi Manabu,Kayesh Mohammad Enamul Hoque,Kohara Michinori,Tsukiyama-Kohara Kyoko,Harashima Hideyoshi
Journal of controlled release : official journal of the Controlled Release Society
The clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) system has considerable therapeutic potential for use in treating a wide range of intractable genetic and infectious diseases including hepatitis B virus (HBV) infections. While non-viral delivery technologies for the CRISPR/Cas system are expected to have clinical applications, difficulties associated with the clinically relevant synthesis of formulations and the poor efficiency of delivery severely hinder therapeutic genome editing. We report herein on the production of a lipid nanoparticle (LNP)-based CRISPR/Cas ribonucleoprotein (RNP) delivery nanoplatform synthesized using a clinically relevant mixer-equipped microfluidic device. DNA cleavage activity and the aggregation of Cas enzymes was completely avoided under the optimized synthetic conditions. The optimized formulation, which was identified through 2 steps of design of experiments, exhibited excellent gene disruption (up to 97%) and base substitution (up to 23%) without any apparent cytotoxicity. The addition of negative charges to the RNPs by complexing single-stranded oligonucleotide (ssON) significantly enhanced the delivery of both Cas9 and Cpf1 RNPs. The optimized formulation significantly suppressed both HBV DNA and covalently closed circular DNA (cccDNA) in HBV-infected human liver cells compared to adeno-associated virus type 2 (AAV2). These findings represent a significant contribution to the development of CRISPR/Cas RNP delivery technology and its practical applications in genome editing therapy.
10.1016/j.jconrel.2020.12.013
Microfluidic assembly of "Turtle-Like" shaped solid lipid nanoparticles for lysozyme delivery.
International journal of pharmaceutics
After two decades of research in the field of nanomedicine, nanoscale delivery systems for biologicals are becoming clinically relevant tools. Microfluidic-based fabrication processes are replacing conventional techniques based on precipitation, emulsion, and homogenization. Here, the focus is on solid lipid nanoparticles (SLNs) for the encapsulation and delivery of lysozyme (LZ) as a model biologic. A thorough analysis was conducted to compare conventional versus microfluidic-based production techniques, using a 3D-printed device. The efficiency of the microfluidic technique in producing LZ-loaded SLNs (LZ SLNs) was demonstrated: LZ SLNs were found to have a lower size (158.05 ± 4.86 nm vs 180.21 ± 7.46 nm) and higher encapsulation efficacy (70.15 ± 1.65 % vs 53.58 ± 1.13 %) as compared to particles obtained with conventional methods. Cryo-EM studies highlighted a peculiar turtle-like structure on the surface of LZ SLNs. In vitro studies demonstrated that LZ SLNs were suitable to achieve a sustained release over time (7 days). Enzymatic activity of LZ entrapped into SLNs was challenged on Micrococcus lysodeikticus cultures, confirming the stability and potency of the biologic. This systematic analysis demonstrates that microfluidic production of SLNs can be efficiently used for encapsulation and delivery of complex biological molecules.
10.1016/j.ijpharm.2022.122479
Heptapeptide-loaded solid lipid nanoparticles for cosmetic anti-aging applications.
Suter Franz,Schmid Daniel,Wandrey Franziska,Zülli Fred
European journal of pharmaceutics and biopharmaceutics : official journal of Arbeitsgemeinschaft fur Pharmazeutische Verfahrenstechnik e.V
The cosmetic industry requires more and more expensive actives and ingredients such as retinol, coenzyme Q10, proteins, peptides and biotechnologically produced molecules. In this study, we demonstrate the development of a cost effective formulation of a nanostructured lipid carrier (NLC) or solid lipid nanoparticles (SLN) improving peptide delivery into skin. NLC or SLN are very suitable vehicles for the delivery of active ingredients into skin. The SLN, produced by using hot high pressure homogenization method combine advantages such as physical stability, protection of incorporated labile actives and controlled release. By the used method we dispersed the amorphous heptapeptide DEETGEF in shea butter and homogenized this pre-dispersion at 60°C together with the water phase using a Microfluidizer at 1000bar. The analysis of the obtained SLN-P7 showed a particle size of 173nm, incorporated peptide of 0.014%, entrapment efficiency of 90.8%, melting peak (DSC) of the core lipid of 27°C and a zeta potential of -54mV. By an ex vivo study with skin explants we could stimulate NQO1 (NAD(P)H quinone oxidoreductase), HMOX1 (Heme oxygenase-1) and PRDX1 (Peroxiredoxin-1) genes all of which are cell protecting enzymes. In a multicellular protection against UV induced stress study with skin explants we detected the formation of sun burn cells and the number and morphology of Langerhans cells. The application of our SLN-P7 formulation on skin explants led to a significant and dose dependent protection against UV irradiation. In the clinical suction blister study, irradiation with UVA light for two hours after final product application led to a statistically significant increase of the 8-OhdG (8-hydroxy-2'-deoxyguanosine) concentration in the human epidermis. The skin treated with our verum formulation showed a statistically significant 20% decrease in DNA damage compared to placebo. In conclusion, it was demonstrated that SLN technology enabled peptide delivery into skin allowing it to perform protective functions.
10.1016/j.ejpb.2016.06.014
One-step microfluidics production of enzyme-loaded liposomes for the treatment of inflammatory diseases.
Costa Clarinda,Liu Zehua,Simões Sandra I,Correia Alexandra,Rahikkala Antti,Seitsonen Jani,Ruokolainen Janne,Aguiar-Ricardo Ana,Santos Hélder A,Corvo M Luísa
Colloids and surfaces. B, Biointerfaces
The biopharmaceuticals market is constantly growing. Despite their advantages over the conventional drugs, biopharmaceuticals have short biological half-lifes, which can be increased using liposomes. However, the common bulk methods to produce biopharmaceuticals-loaded liposomes result in lost of encapsulation efficiency (E.E.), resulting in an expensive process. Herein, the encapsulation of a therapeutic enzyme in liposomes is proposed, using a glass-capillary microfluidic technique. Cu,Zn- Superoxide dismutase (SOD) is successfully encapsulated into liposomes (SOD@Liposomes). SOD@Liposomes with a mean size of 135 ± 41 nm, a polydispersity index of 0.13 ± 0.01, an E.E. of 59 ± 6 % and an enzyme activity of 82 ± 3 % are obtained. in vivo experiments show, through an ear edema model, that SOD@Liposomes administered by the intravenous route enable an edema inhibition of 65 % ± 8 %, over the 20 % ± 13 % of SOD in its free form. The histopathological analyses show a higher inflammatory cell accumulation on the ear treated with SOD in its free form, than treated with SOD@Liposomes. Overall, this work highlights the potential of microfluidics for the production of enzyme-loaded liposomes with high encapsulation efficiency, with the intrinsic advantages of the low time-consuming and easily upscaling microfluidic assembly method.
10.1016/j.colsurfb.2020.111556