Evaluating the impact of experimental shear flow parameters on nanoparticle protein corona formation

The use of nanoparticles has increasingly been implemented in biomedical applications including the diagnosis and treatment of disease. When administered to a biological system, nanoparticles spontaneously interact with their surrounding environment, leading to the surface-adsorption of small molecules and biomacromolecules. The protein component of the surface-adsorbed species, is often referred to as the "protein corona". The composition of the protein corona is governed by nanoparticle properties, incubation media and parameters related to the environment in which nanoparticle incubations are performed. In this study, we investigated the formation of protein corona on polystyrene nanoparticles which have different surface chemistries and the impact of experimental incubation parameters, including centrifugation-resuspension protocols, incubation duration and shear flow rate conditions. The particle characteristics measured include size distribution, zeta potential and total protein content. Our findings show significant differences in nanoparticle size following exposure to media containing proteins across the three different nanoparticle surface chemistries. These findings were also confirmed by total protein concentration measurements performed on nanoparticles recovered from bulk media, and measurements of the composition of surface-adsorbed proteins by gel electrophoresis. We also found that exposure to different shear flow conditions alters both the thickness and the composition of surface-adsorbed protein coronas. In parallel to analysis of nanoparticles isolated using the centrifugation-resuspension protocol, we performed in situ analysis of nanoparticle size in media containing proteins. Results obtained from these measurements highlight that the recovery procedure is disruptive to the protein corona and therefore the need for investigative methods that do not alter the properties of the nanoparticle coronas. Nanomedicines are generally intended for administration via injection, and our findings show that parameters such as shear flow and media composition can significantly alter nanoparticle physicochemical parameters. Overall, we show that the recovery protocol can significantly alter particle parameters in addition to the overall protein composition of surface-adsorbed proteins. We recommend that nanoparticle characterization pipelines studying bio-nano interactions during early nanomedicine development consider experimental design in the context of biologically-relevant shear flow conditions and media composition because these parameters can significantly alter particle physical parameters and the subsequent conclusions drawn from such studies

Nanoparticle isolation from biological media for protein corona analysis : the impact of incubation and recovery protocols on nanoparticle properties

Nanoparticles are increasingly implemented in biomedical applications, including the diagnosis and treatment of disease. When exposed to complex biological media, nanoparticles spontaneously interact with their surrounding environment, leading to the surface-adsorption of small and bio- macromolecules- termed the "corona". Corona composition is governed by nanoparticle properties and incubation parameters. While the focus of most studies is on the protein signature of the nanoparticle corona, the impact of experimental protocols on nanoparticle size in the presence of complex biological media, and the impact of nanoparticle recovery from biological media has not yet been reported. Here using a non-degradable robust model, we show how centrifugation-resuspension protocols used for the isolation of nanoparticles from incubation media, incubation duration and shear flow conditions alter nanoparticle parameters including particle size, zeta potential and total protein content. Our results show significant changes in nanoparticle size following exposure to media containing protein under different flow conditions, which also altered the composition of surface-adsorbed proteins profiled by SDS-PAGE. Our in situ analysis of nanoparticle size in media containing protein using particle tracking analysis highlights that centrifugation-resuspension is disruptive to agglomerates that are spontaneously formed in protein containing media, highlighting the need for in situ analytical methods that do not alter the intermediates formed following nanoparticle exposure to biological media. Nanomedicines are mostly intended for parenteral administration, and our findings show that parameters such as shear flow can significantly alter nanoparticle physicochemical parameters. Overall, we show that the centrifugation-resuspension isolation of nanoparticles from media significantly alters particle parameters in addition to the overall protein composition of surface-adsorbed proteins. We recommend that nanoparticle characterization pipelines studying bio-nano interactions during early nanomedicine development consider biologically-relevant shear flow conditions and media composition that can significantly alter particle physical parameters and subsequent conclusions from these studies

Chemical and antimicrobial profiling of propolis from different regions within Libya.

Extracts from twelve samples of propolis collected from different regions of Libya were tested for their activity against Trypanosoma brucei, Leishmania donovani, Plasmodium falciparum, Crithidia fasciculata and Mycobacterium marinum and the cytotoxicity of the extracts was tested against mammalian cells. All the extracts were active to some degree against all of the protozoa and the mycobacterium, exhibiting a range of EC50 values between 1.65 and 53.6 μg/ml. The toxicity against mammalian cell lines was only moderate; the most active extract against the protozoan species, P2, displayed an IC50 value of 53.2 μg/ml. The extracts were profiled by using liquid chromatography coupled to high resolution mass spectrometry. The data sets were extracted using m/z Mine and the accurate masses of the features extracted were searched against the Dictionary of Natural Products (DNP). A principal component analysis (PCA) model was constructed which, in combination with hierarchical cluster analysis (HCA), divided the samples into five groups. The outlying groups had different sets of dominant compounds in the extracts, which could be characterised by their elemental composition. Orthogonal partial least squares (OPLS) analysis was used to link the activity of each extract against the different micro-organisms to particular components in the extracts

Studies on development of a Leishmanial vaccine

Leishmaniasis is a disease caused by infection with the obligate intracellular parasite of the genus Leishmania. Leishmaniasis is a public health problem; it is estimated that approximately 12-15 million people worldwide are infected. An estimated 1.5-2 million new cases occur each year, 350 million people are at risk of infection, and it causes 70000 deaths per year.;Therefore, development of a vaccine to prevent infection is required. The overall aim of this study was to develop a vaccine to protect against Leishmania infection using live nonpathogenic L. tarentolae transfected with gamma glutamylcysteine synthetase (γGCS) from three different species (L. donovani, L. mexicana and L. major).;Previous studies for expressing recombinant γGCS using E. coli as an expression system have shown that it was not possible to produce pure full-length recombinant γGCS protein. Therefore, in this project, using an expression vector that is phylogenically more like Leishmania. L. tarentolae has been used as an expression system for eukayotic recombinant proteins.;Studies of the expression of L. donovani, L. mexicana and L. major γGCS recombinant proteins showed that parasites integrated with a green fluorescent protein γGCS-His gene gave stable expression of the fusion protein for six months without the requirement for antibiotics to maintain expression of the gene insert. Supplementing the culture medium with hydrogen peroxide (H2O2) increased proliferation of parasites in cell culture.;The addition of a 12 μM of H2O2 supplement increased the number of the parasites in cell culture, which can conclude that expression of the GFP-γGCS-His recombinant protein has been increased.;Expression of the recombinant GFP-γGCS-His protein produced full-length protein and truncated protein, but after isolation from an affinity column, it was impossible to produce pure full-length protein for all three transfected parasites. Therefore, this purification method failed to remove non-specific protein contamination.;The live non-pathogenic lizard parasite, L. tarentolae, expressing elected Leishmania antigens has recently provided a promising new approach as a safe and effective live vaccine candidate to prevent Leishmaniasis. Here, this study evaluated the immunoprotective potential of a live vaccine against L. major infection in BALB/c mice, using L. tarentolae transfected with the GFP-γGCS-His sequence gene from one of three different species (L. donovani, L. major or L. mexicana) or a 'triple vaccine' using a 1:1:1 mixture of L. tarentolae transfected with the GFP-γGCS-His sequence gene of the three pathogenic species;Vaccination with transfected L. tarentolae with GFP-γGCS-His gene from L. donovani, L. major and L. mexicana (triple vaccine) induced significant parasite specific Th1 immune responses based on antibody titres and cytokine production in vitro in stimulated splenocytes and popliteal lymph nodes from immunised mice.;Vaccination by subcutaneous injection with the triple vaccine caused the highest percentage reduction in parasite burdens compared to controls ± SE, was 94% ± 0.01 in L. major infected mice. Vaccination with L.t L. maj GFP-γGCS-His, L. mex GFP-γGCS-His and L. don GFP-γGCS-His parasites failed to give significant protection against L. major infection, but vaccination with L. t L. maj GFP-γGCSHis resulted in an 86% ± 0.01 suppression in parasite burden compared to controls.;In conclusion, the results of this study indicate that vaccination with transfected L. tarentolae parasites against L. major infection enhanced the protective efficacy and that the triple vaccine is a potential vaccine candidate.Leishmaniasis is a disease caused by infection with the obligate intracellular parasite of the genus Leishmania. Leishmaniasis is a public health problem; it is estimated that approximately 12-15 million people worldwide are infected. An estimated 1.5-2 million new cases occur each year, 350 million people are at risk of infection, and it causes 70000 deaths per year.;Therefore, development of a vaccine to prevent infection is required. The overall aim of this study was to develop a vaccine to protect against Leishmania infection using live nonpathogenic L. tarentolae transfected with gamma glutamylcysteine synthetase (γGCS) from three different species (L. donovani, L. mexicana and L. major).;Previous studies for expressing recombinant γGCS using E. coli as an expression system have shown that it was not possible to produce pure full-length recombinant γGCS protein. Therefore, in this project, using an expression vector that is phylogenically more like Leishmania. L. tarentolae has been used as an expression system for eukayotic recombinant proteins.;Studies of the expression of L. donovani, L. mexicana and L. major γGCS recombinant proteins showed that parasites integrated with a green fluorescent protein γGCS-His gene gave stable expression of the fusion protein for six months without the requirement for antibiotics to maintain expression of the gene insert. Supplementing the culture medium with hydrogen peroxide (H2O2) increased proliferation of parasites in cell culture.;The addition of a 12 μM of H2O2 supplement increased the number of the parasites in cell culture, which can conclude that expression of the GFP-γGCS-His recombinant protein has been increased.;Expression of the recombinant GFP-γGCS-His protein produced full-length protein and truncated protein, but after isolation from an affinity column, it was impossible to produce pure full-length protein for all three transfected parasites. Therefore, this purification method failed to remove non-specific protein contamination.;The live non-pathogenic lizard parasite, L. tarentolae, expressing elected Leishmania antigens has recently provided a promising new approach as a safe and effective live vaccine candidate to prevent Leishmaniasis. Here, this study evaluated the immunoprotective potential of a live vaccine against L. major infection in BALB/c mice, using L. tarentolae transfected with the GFP-γGCS-His sequence gene from one of three different species (L. donovani, L. major or L. mexicana) or a 'triple vaccine' using a 1:1:1 mixture of L. tarentolae transfected with the GFP-γGCS-His sequence gene of the three pathogenic species;Vaccination with transfected L. tarentolae with GFP-γGCS-His gene from L. donovani, L. major and L. mexicana (triple vaccine) induced significant parasite specific Th1 immune responses based on antibody titres and cytokine production in vitro in stimulated splenocytes and popliteal lymph nodes from immunised mice.;Vaccination by subcutaneous injection with the triple vaccine caused the highest percentage reduction in parasite burdens compared to controls ± SE, was 94% ± 0.01 in L. major infected mice. Vaccination with L.t L. maj GFP-γGCS-His, L. mex GFP-γGCS-His and L. don GFP-γGCS-His parasites failed to give significant protection against L. major infection, but vaccination with L. t L. maj GFP-γGCSHis resulted in an 86% ± 0.01 suppression in parasite burden compared to controls.;In conclusion, the results of this study indicate that vaccination with transfected L. tarentolae parasites against L. major infection enhanced the protective efficacy and that the triple vaccine is a potential vaccine candidate

Production of mRNA lipid nanoparticles using advanced crossflow micromixing

Lipid nanoparticles (LNPs) play a crucial role in RNA-based therapies, and their production is generally based on nanoprecipitation and coalescence of lipids around an RNA core. This process is primarily controlled via the rate of ethanol-lipid to mRNA aqueous phase mixing. When considering a manufacturing process for LNPs, choosing between single-use and reusable systems influences production efficiency, cost, and sustainability. Single-use systems offer advantages in minimising contamination and downtime, while reusable systems reduce plastic use, avoid the problem of leachables, especially in solvents and present long-term cost savings. Our results demonstrate the reproducible production of mRNA-LNPs with controlled Critical Quality Attributes, including high mRNA encapsulation from the initial screening scale through to GMP-scale production, where the same mixing ratio can be adopted across all product speeds from 30 to 500 mL/min used. These mRNA-LNPs were confirmed to be effective as therapeutics (protein expression) and for vaccination (antibody and cytokine responses)

Immunisation with transgenic L. tarentolae expressing gamma glutamyl cysteine synthetase from pathogenic Leishmania species protected against L. major and L. donovani infection in a murine model

Leishmaniasis is a protozoan disease responsible for significant morbidity and mortality. There is no recommended vaccine to protect against infection. In this study, transgenic Leishmania tarentolae expressing gamma glutamyl cysteine synthetase (γGCS) from three pathogenic species were produced and their ability to protect against infection determined using models of cutaneous and visceral leishmaniasis. The ability of IL-2-producing PODS® to act as an adjuvant was also determined in L. donovani studies. Two doses of the live vaccine caused a significant reduction in L. major (p < 0.001) and L. donovani (p < 0.05) parasite burdens compared to their respective controls. In contrast, immunisation with wild type L. tarentolae, using the same immunisation protocol, had no effect on parasite burdens compared to infection controls. Joint treatment with IL-2-producing PODS® enhanced the protective effect of the live vaccine in L. donovani studies. Protection was associated with a Th1 response in L. major and a mixed Th1/Th2 response in L. donovani, based on specific IgG1 and IgG2a antibody and cytokine production from in vitro proliferation assays using antigen-stimulated splenocytes. The results of this study provide further proof that γGCS should be considered a candidate vaccine for leishmaniasis

Examination of the effect of niosome preparation methods in encapsulating model antigens on the vesicle characteristics and their ability to induce immune responses

Niosome nanoparticles can be prepared using different methods, each of which can affect the size and homogeneity of the prepared particles. The aim of this study was to establish if the method of preparation impacted on the prepared vesicles when loaded with a model protein and the type of immune responses induced to the vaccine antigen. Niosomes were prepared using both the traditional thin film hydration (TFH) technique and the microfluidic mixing (MM) technique. Influenza antigen was then entrapped in the niosomes and formulations tested for their ability to induce in vivo immune responses in immunised BALB/c mice. Niosomes prepared by MM had a mean size of 157 ± 1.8 nm and were significantly more uniform compared with the niosomes prepared using TFH (mean size 388 ± 10 nm). Niosomes play a key role as an adjuvant to help raise high antibody immune responses. This was confirmed in this study since animals treated with both types of niosomes and antigen were more responsive than unentrapped (free) antigen. Cytokine analysis showed that the TFH niosomes induced a Th1 immune response by raising IgG2a and high levels of IFN-ɣ, while the MM niosomes induced a Th2 immune response by inducing IgG1 (p <.05). These results confirmed that the method of preparation of the niosomes nanoparticles induced different immune responses and the average particle size of the niosomes differed depending on the method of manufacture. This indicates that particle size and uniformity are of importance and should be taken into consideration when designing an oral based delivery system for vaccine delivery

Libyan map showing the collection points Libyan Propolis samples P1 (Alagoria), P2 (Gaminis), P3 (Byda), P4 (Quba), P5,P6, P7 (Kufra), P8(Ghadames), P9 (Tripoli), P10 (Khasr Khiar), P11, P12 (Khumas).

<p>Libyan map showing the collection points Libyan Propolis samples P1 (Alagoria), P2 (Gaminis), P3 (Byda), P4 (Quba), P5,P6, P7 (Kufra), P8(Ghadames), P9 (Tripoli), P10 (Khasr Khiar), P11, P12 (Khumas).</p

Activity of samples P1-P12 against <i>P</i>.<i>falciparum</i> (n = 3).

<p>Activity of samples P1-P12 against <i>P</i>.<i>falciparum</i> (n = 3).</p