Understanding protein corona formation under shear flow conditions

Nanoparticles are small colloidal particles in the 1-100 nm size range. Polymeric nanoparticles are routinely explored for the development of novel drug delivery systems due to their unique and customizable physical and chemical characteristics. Upon introduction to protein-containing medium, nanoparticles will spontaneously adsorb proteins onto their surface and form what is known as the ‘protein corona’. The protein corona leads to changes in the physical and chemical parameters of nanoparticles, which subsequently alters their biological fate (cellular uptake, biodistribution). With most nanoparticles intended for intravenous administration, it is crucial to understand the impact of biological shear flow conditions on protein corona formation and how the protein corona influences their colloidal stability

The development of a novel pipeline for polymeric nanoparticle characterization following incubation under shear flow conditions

Nanoparticles are small colloidal particles with a dimension between 1-100 nm in size. Polymeric nanoparticles are routinely explored for the development of novel medicines for unmet clinical need due to their unique properties. However, there is currently a high attrition rate for bench-clinic translation, and this may be due to a lack of understanding of the behaviour of nanoparticles under physiologically-relevant conditions. Upon administration to protein-containing medium, nanoparticles will spontaneously adsorb proteins onto their surface and form what is known as the ‘protein corona’ (figure 1) . Protein corona formation leads to changes in the physical and chemical parameters of nanoparticles, which subsequently alters their biological fate (cellular uptake, biodistribution). With most nanoparticles intended for intravenous administration, it is therefore crucial to characterize the impact of biological shear flow conditions on nanoparticle-protein interactions and how this impact their colloidal stability. Aims & Objectives: 1)To develop a robust pipeline for the reproducible characterization of nanoparticles following protein corona formation under physiologically relevant shear flow conditions. 2) To use a range of nanoparticle isolation and analytical techniques to measure the impact of sample handling conditions on particle parameters using model nanoparticles (polystyrene latex)

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

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

A roadmap for patient-public involvement and engagement (PPIE) : recounting the untold stories of breast cancer patient experiences

Introduction Breast cancer remains a prevalent disease in women worldwide. Though significant advancements in the standard of care for breast cancer have contributed to improved patient survival and quality of life, a breast cancer diagnosis and subsequent treatment interventions have a long-lasting impact on patients’ lived experiences. A high-quality healthcare system uses a patient-centred approach to healthcare, with patient engagement being a central pillar in the delivery of patient-centred care. However, the disconnect between patients and researchers can translate into research lacking real-world relevance to patient health needs. Here, we report a patient and stakeholder engagement workshop series that was conceptualized with the goal of promoting dialogue between patients with breast cancer, breast cancer researchers and the clinician involved in their care. We present the collaborative learning process and emerging opportunities from this patient engagement workshop series as a community-academic partnership. Method We report on a three-part storytelling workshop, with the scope of the workshops including topics related to raising awareness of the patient lived experience following a breast cancer diagnosis, breast cancer research activities undertaken by researchers, and the approach used by multidisciplinary healthcare teams in the management of breast cancer using storytelling as a tool. We used an iterative approach to cohort trust and relationship building, narrative development, and the use of multiple media formats to capture patient stories. This included the use of object memories, storytelling prompt cards and open-mic audio format to capture patient stories from diagnosis to treatment, and remission. Results 20 patients shared their stories with key themes emerging from the qualitative analysis of audio recordings. For many, this was the first time they had spoken about their breast cancer experience beyond family and friends. Emerging themes included common public misconceptions about a breast cancer diagnosis, the importance of self-advocacy in patient decision making about treatment, and the complex emotional journey experienced by patients diagnosed with breast cancer. The group-based storytelling approach provided collective empowerment to share personal experiences and connect meaningfully across the peer community. Conclusion While a breast cancer diagnosis can be overwhelming from a physical, social, emotional and cognitive perspective, storytelling as a patient engagement approach can build patient trust in researchers, ensuring that as key stakeholders they are involved in the process of research. Understanding the patient perspective of a breast cancer diagnosis and subsequent experiences can support healthcare professionals in developing an empathetic approach to sharing information, and involving patients in shared decision making about their healthcare

Characterization of polymeric nanoparticle-protein interactions

Polymeric nanoparticles have shown significant potential for the development of new medicines for unmet clinical need. Upon administration into protein-containing media, proteins will spontaneously adsorb onto the surface of nanoparticles and form a 'protein corona'. Protein corona formation alters the physiochemical properties of nanoparticles which consequently impacts their biological fate. The aim of this study was to develop a robust pipeline for the reproducible characterization of polymeric nanoparticles following protein corona formation

Investigating the impact of shear flow on nanoparticle-protein interactions

Most nanoparticle-based therapies are intended forintravenous administration, exposing them to associated hemodynamic parametersand the presence of cells and biomacromolecules post-administration. While mostefforts in nanomedicine development focus on formulation stability, the rangeof biologically-relevant approaches probing nanoparticle stability inbiological media remain limited in scope. In the present study, we examine therole of surface chemistry in nanoparticle-protein interactions using threepolystyrene latex nanoparticle chemistries. These nanoparticles were treated inmedia mimicking cell culture conditions, and the impact of static co-incubationsversus flow on nanoparticle parameters were compared. Following treatment withprotein-containing media, we performed analysis of nanoparticle parameters usingeither the centrifugation-wash step or in-situ analyses to compare the effectsof isolation protocols on nanoparticle physicochemical parameters. Overall, ourfindings show that flow and sample recovery methods significantly impacted the concentrationand composition of surface-adsorbed proteins. Amine-modified latex nanoparticlesshowed the most pronounced susceptibility to flow and nanoparticle isolationtechniques. The implications of this work lie in the development of more biologically-relevantand harmonized approaches in measuring the nanoparticle protein corona, sincesample preparation techniques and analytical approaches used, may impact the translationalscope and relevance of assays used to measure nanoparticle interactions withbiological media

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

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