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Modern methods of molecular diagnostics and therapy have revolutionized the field of medicine in recent years by providing more precise and effective tools for detecting and treating diseases. This progress includes a growing exploration of the body's secreted vesicles, known as extracellular vesicles (EVs), for both diagnostic and therapeutic purposes. EVs are a heterogeneous population of lipid bilayer vesicles secreted by almost every cell type studied so far. They are detected in body fluids and conditioned culture media from living cells. EVs play a crucial role in communication between cells and organs, both locally and over long distances. They are recognized for their ability to transport endogenous RNA and proteins between cells, including messenger RNA (mRNA), microRNA (miRNA), misfolded neurodegenerative proteins, and several other biomolecules. This review explores the dual utilization of EVs, serving not only for diagnostic purposes but also as a platform for delivering therapeutic molecules to cells and tissues. Through an exploration of their composition, biogenesis, and selective cargo packaging, we elucidate the intricate mechanisms behind RNA transport between cells via EVs, highlighting their potential use for both diagnostic and therapeutic applications. Finally, it addresses challenges and outlines prospective directions for the clinical utilization of EVs., CC BY 4.0Corresponding author: Hadi ValadiE-mail address: hadi.valadi@gu.se (H. Valadi).This work has been supported by grants from the Swedish Foundation of Strategic Research (Stiftelsen för strategisk forskning: SSF) in the Industrial Research Centre, FoRmulaEx – Nucleotide Functional Drug Delivery (Grant ID: IRC15-0065), the Swedish research council (VR, Grant ID: 2020-01316), and The Swedish Knowledge Foundation (KKS, Grant ID: 2020-0014).

Modern methods of molecular diagnostics and therapy have revolutionized the field of medicine in recent years by providing more precise and effective tools for detecting and treating diseases. This progress includes a growing exploration of the body's secreted vesicles, known as extracellular vesicles (EVs), for both diagnostic and therapeutic purposes. EVs are a heterogeneous population of lipid bilayer vesicles secreted by almost every cell type studied so far. They are detected in body fluids and conditioned culture media from living cells. EVs play a crucial role in communication between cells and organs, both locally and over long distances. They are recognized for their ability to transport endogenous RNA and proteins between cells, including messenger RNA (mRNA), microRNA (miRNA), misfolded neurodegenerative proteins, and several other biomolecules. This review explores the dual utilization of EVs, serving not only for diagnostic purposes but also as a platform for delivering therapeutic molecules to cells and tissues. Through an exploration of their composition, biogenesis, and selective cargo packaging, we elucidate the intricate mechanisms behind RNA transport between cells via EVs, highlighting their potential use for both diagnostic and therapeutic applications. Finally, it addresses challenges and outlines prospective directions for the clinical utilization of EVs., CC BY 4.0Corresponding author: Hadi ValadiE-mail address: hadi.valadi@gu.se (H. Valadi).This work has been supported by grants from the Swedish Foundation of Strategic Research (Stiftelsen för strategisk forskning: SSF) in the Industrial Research Centre, FoRmulaEx – Nucleotide Functional Drug Delivery (Grant ID: IRC15-0065), the Swedish research council (VR, Grant ID: 2020-01316), and The Swedish Knowledge Foundation (KKS, Grant ID: 2020-0014).

Modern methods of molecular diagnostics and therapy have revolutionized the field of medicine in recent years by providing more precise and effective tools for detecting and treating diseases. This progress includes a growing exploration of the body's secreted vesicles, known as extracellular vesicles (EVs), for both diagnostic and therapeutic purposes. EVs are a heterogeneous population of lipid bilayer vesicles secreted by almost every cell type studied so far. They are detected in body fluids and conditioned culture media from living cells. EVs play a crucial role in communication between cells and organs, both locally and over long distances. They are recognized for their ability to transport endogenous RNA and proteins between cells, including messenger RNA (mRNA), microRNA (miRNA), misfolded neurodegenerative proteins, and several other biomolecules. This review explores the dual utilization of EVs, serving not only for diagnostic purposes but also as a platform for delivering therapeutic molecules to cells and tissues. Through an exploration of their composition, biogenesis, and selective cargo packaging, we elucidate the intricate mechanisms behind RNA transport between cells via EVs, highlighting their potential use for both diagnostic and therapeutic applications. Finally, it addresses challenges and outlines prospective directions for the clinical utilization of EVs., CC BY 4.0Corresponding author: Hadi ValadiE-mail address: hadi.valadi@gu.se (H. Valadi).This work has been supported by grants from the Swedish Foundation of Strategic Research (Stiftelsen för strategisk forskning: SSF) in the Industrial Research Centre, FoRmulaEx – Nucleotide Functional Drug Delivery (Grant ID: IRC15-0065), the Swedish research council (VR, Grant ID: 2020-01316), and The Swedish Knowledge Foundation (KKS, Grant ID: 2020-0014).

Modern methods of molecular diagnostics and therapy have revolutionized the field of medicine in recent years by providing more precise and effective tools for detecting and treating diseases. This progress includes a growing exploration of the body's secreted vesicles, known as extracellular vesicles (EVs), for both diagnostic and therapeutic purposes. EVs are a heterogeneous population of lipid bilayer vesicles secreted by almost every cell type studied so far. They are detected in body fluids and conditioned culture media from living cells. EVs play a crucial role in communication between cells and organs, both locally and over long distances. They are recognized for their ability to transport endogenous RNA and proteins between cells, including messenger RNA (mRNA), microRNA (miRNA), misfolded neurodegenerative proteins, and several other biomolecules. This review explores the dual utilization of EVs, serving not only for diagnostic purposes but also as a platform for delivering therapeutic molecules to cells and tissues. Through an exploration of their composition, biogenesis, and selective cargo packaging, we elucidate the intricate mechanisms behind RNA transport between cells via EVs, highlighting their potential use for both diagnostic and therapeutic applications. Finally, it addresses challenges and outlines prospective directions for the clinical utilization of EVs., CC BY 4.0Corresponding author: Hadi ValadiE-mail address: hadi.valadi@gu.se (H. Valadi).This work has been supported by grants from the Swedish Foundation of Strategic Research (Stiftelsen för strategisk forskning: SSF) in the Industrial Research Centre, FoRmulaEx – Nucleotide Functional Drug Delivery (Grant ID: IRC15-0065), the Swedish research council (VR, Grant ID: 2020-01316), and The Swedish Knowledge Foundation (KKS, Grant ID: 2020-0014).

Modern methods of molecular diagnostics and therapy have revolutionized the field of medicine in recent years by providing more precise and effective tools for detecting and treating diseases. This progress includes a growing exploration of the body's secreted vesicles, known as extracellular vesicles (EVs), for both diagnostic and therapeutic purposes. EVs are a heterogeneous population of lipid bilayer vesicles secreted by almost every cell type studied so far. They are detected in body fluids and conditioned culture media from living cells. EVs play a crucial role in communication between cells and organs, both locally and over long distances. They are recognized for their ability to transport endogenous RNA and proteins between cells, including messenger RNA (mRNA), microRNA (miRNA), misfolded neurodegenerative proteins, and several other biomolecules. This review explores the dual utilization of EVs, serving not only for diagnostic purposes but also as a platform for delivering therapeutic molecules to cells and tissues. Through an exploration of their composition, biogenesis, and selective cargo packaging, we elucidate the intricate mechanisms behind RNA transport between cells via EVs, highlighting their potential use for both diagnostic and therapeutic applications. Finally, it addresses challenges and outlines prospective directions for the clinical utilization of EVs., CC BY 4.0Corresponding author: Hadi ValadiE-mail address: hadi.valadi@gu.se (H. Valadi).This work has been supported by grants from the Swedish Foundation of Strategic Research (Stiftelsen för strategisk forskning: SSF) in the Industrial Research Centre, FoRmulaEx – Nucleotide Functional Drug Delivery (Grant ID: IRC15-0065), the Swedish research council (VR, Grant ID: 2020-01316), and The Swedish Knowledge Foundation (KKS, Grant ID: 2020-0014).

Lipid nanoparticles (LNPs) are currently used to transport functional mRNAs, such as COVID-19 mRNA vaccines. The delivery of angiogenic molecules, such as therapeutic VEGF-A mRNA, to ischemic tissues for producing new blood vessels is an emerging strategy for the treatment of cardiovascular diseases. Here, the authors deliver VEGF-A mRNA via LNPs and study stoichiometric quantification of their uptake kinetics and how the transport of exogenous LNP-mRNAs between cells is functionally extended by cells’ own vehicles called extracellular vesicles (EVs). The results show that cellular uptake of LNPs and their mRNA molecules occurs quickly, and that the translation of exogenously delivered mRNA begins immediately. Following the VEGF-A mRNA delivery to cells via LNPs, a fraction of internalized VEGF-A mRNA is secreted via EVs. The overexpressed VEGF-A mRNA is detected in EVs secreted from three different cell types. Additionally, RNA-Seq analysis reveals that as cells’ response to LNP-VEGF-A mRNA treatment, several overexpressed proangiogenic transcripts are packaged into EVs. EVs are further deployed to deliver VEGF-A mRNA in vitro and in vivo. Upon equal amount of VEGF-A mRNA delivery via three EV types or LNPs in vitro, EVs from cardiac progenitor cells are the most efficient in promoting angiogenesis per amount of VEGF-A protein produced. Intravenous administration of luciferase mRNA shows that EVs could distribute translatable mRNA to different organs with the highest amounts of luciferase detected in the liver. Direct injections of VEGF-A mRNA (via EVs or LNPs) into mice heart result in locally produced VEGF-A protein without spillover to liver and circulation. In addition, EVs from cardiac progenitor cells cause minimal production of inflammatory cytokines in cardiac tissue compared with all other treatment types. Collectively, the data demonstrate that LNPs transform EVs as functional extensions to distribute therapeutic mRNA between cells, where EVs deliver this, CC BY 4.0© 2023 The Authors. Advanced Science published by Wiley-VCH GmbH.E-mail: hadi.valadi@gu.seThe authors acknowledge the support from the National Genomics Infrastructure in Stockholm funded by Science for Life (SciLife) Laboratory, the Knut and Alice Wallenberg Foundation and the Swedish Research Council, and SNIC/Uppsala Multidisciplinary Center for Advanced Computational Science for assistance with massively parallel sequencing and access to the UPPMAX computational infrastructure. Moreover, the authors acknowledge Mr. Mario Soriano Navarro at the Responsable Servicio Microscopía Electrónica, Valencia Spain, for technical assistance. This work was supported by grants from the Swedish Foundation of Strategic Research (Stiftelsen för strategisk forskning: SSF) in the Industrial Research Centre, FoRmulaEx - Nucleotide Functional Drug Delivery (Grant ID: IRC15-0065), the Swedish research council (VR, Grant ID: 2020-01316), and the Swedish governmental agency for innovation systems (VINNOVA, Grant ID: 2017-02960). This research was also funded by the Systems Biology Research Centre at the University of Skövde under grants from the Knowledge Foundation (Grant ID: 20160330).

Lipid nanoparticles (LNPs) are currently used to transport functional mRNAs, such as COVID-19 mRNA vaccines. The delivery of angiogenic molecules, such as therapeutic VEGF-A mRNA, to ischemic tissues for producing new blood vessels is an emerging strategy for the treatment of cardiovascular diseases. Here, the authors deliver VEGF-A mRNA via LNPs and study stoichiometric quantification of their uptake kinetics and how the transport of exogenous LNP-mRNAs between cells is functionally extended by cells’ own vehicles called extracellular vesicles (EVs). The results show that cellular uptake of LNPs and their mRNA molecules occurs quickly, and that the translation of exogenously delivered mRNA begins immediately. Following the VEGF-A mRNA delivery to cells via LNPs, a fraction of internalized VEGF-A mRNA is secreted via EVs. The overexpressed VEGF-A mRNA is detected in EVs secreted from three different cell types. Additionally, RNA-Seq analysis reveals that as cells’ response to LNP-VEGF-A mRNA treatment, several overexpressed proangiogenic transcripts are packaged into EVs. EVs are further deployed to deliver VEGF-A mRNA in vitro and in vivo. Upon equal amount of VEGF-A mRNA delivery via three EV types or LNPs in vitro, EVs from cardiac progenitor cells are the most efficient in promoting angiogenesis per amount of VEGF-A protein produced. Intravenous administration of luciferase mRNA shows that EVs could distribute translatable mRNA to different organs with the highest amounts of luciferase detected in the liver. Direct injections of VEGF-A mRNA (via EVs or LNPs) into mice heart result in locally produced VEGF-A protein without spillover to liver and circulation. In addition, EVs from cardiac progenitor cells cause minimal production of inflammatory cytokines in cardiac tissue compared with all other treatment types. Collectively, the data demonstrate that LNPs transform EVs as functional extensions to distribute therapeutic mRNA between cells, where EVs deliver this, CC BY 4.0© 2023 The Authors. Advanced Science published by Wiley-VCH GmbH.E-mail: hadi.valadi@gu.seThe authors acknowledge the support from the National Genomics Infrastructure in Stockholm funded by Science for Life (SciLife) Laboratory, the Knut and Alice Wallenberg Foundation and the Swedish Research Council, and SNIC/Uppsala Multidisciplinary Center for Advanced Computational Science for assistance with massively parallel sequencing and access to the UPPMAX computational infrastructure. Moreover, the authors acknowledge Mr. Mario Soriano Navarro at the Responsable Servicio Microscopía Electrónica, Valencia Spain, for technical assistance. This work was supported by grants from the Swedish Foundation of Strategic Research (Stiftelsen för strategisk forskning: SSF) in the Industrial Research Centre, FoRmulaEx - Nucleotide Functional Drug Delivery (Grant ID: IRC15-0065), the Swedish research council (VR, Grant ID: 2020-01316), and the Swedish governmental agency for innovation systems (VINNOVA, Grant ID: 2017-02960). This research was also funded by the Systems Biology Research Centre at the University of Skövde under grants from the Knowledge Foundation (Grant ID: 20160330).

Lipid nanoparticles (LNPs) are currently used to transport functional mRNAs, such as COVID-19 mRNA vaccines. The delivery of angiogenic molecules, such as therapeutic VEGF-A mRNA, to ischemic tissues for producing new blood vessels is an emerging strategy for the treatment of cardiovascular diseases. Here, the authors deliver VEGF-A mRNA via LNPs and study stoichiometric quantification of their uptake kinetics and how the transport of exogenous LNP-mRNAs between cells is functionally extended by cells’ own vehicles called extracellular vesicles (EVs). The results show that cellular uptake of LNPs and their mRNA molecules occurs quickly, and that the translation of exogenously delivered mRNA begins immediately. Following the VEGF-A mRNA delivery to cells via LNPs, a fraction of internalized VEGF-A mRNA is secreted via EVs. The overexpressed VEGF-A mRNA is detected in EVs secreted from three different cell types. Additionally, RNA-Seq analysis reveals that as cells’ response to LNP-VEGF-A mRNA treatment, several overexpressed proangiogenic transcripts are packaged into EVs. EVs are further deployed to deliver VEGF-A mRNA in vitro and in vivo. Upon equal amount of VEGF-A mRNA delivery via three EV types or LNPs in vitro, EVs from cardiac progenitor cells are the most efficient in promoting angiogenesis per amount of VEGF-A protein produced. Intravenous administration of luciferase mRNA shows that EVs could distribute translatable mRNA to different organs with the highest amounts of luciferase detected in the liver. Direct injections of VEGF-A mRNA (via EVs or LNPs) into mice heart result in locally produced VEGF-A protein without spillover to liver and circulation. In addition, EVs from cardiac progenitor cells cause minimal production of inflammatory cytokines in cardiac tissue compared with all other treatment types. Collectively, the data demonstrate that LNPs transform EVs as functional extensions to distribute therapeutic mRNA between cells, where EVs deliver this, CC BY 4.0© 2023 The Authors. Advanced Science published by Wiley-VCH GmbH.E-mail: hadi.valadi@gu.seThe authors acknowledge the support from the National Genomics Infrastructure in Stockholm funded by Science for Life (SciLife) Laboratory, the Knut and Alice Wallenberg Foundation and the Swedish Research Council, and SNIC/Uppsala Multidisciplinary Center for Advanced Computational Science for assistance with massively parallel sequencing and access to the UPPMAX computational infrastructure. Moreover, the authors acknowledge Mr. Mario Soriano Navarro at the Responsable Servicio Microscopía Electrónica, Valencia Spain, for technical assistance. This work was supported by grants from the Swedish Foundation of Strategic Research (Stiftelsen för strategisk forskning: SSF) in the Industrial Research Centre, FoRmulaEx - Nucleotide Functional Drug Delivery (Grant ID: IRC15-0065), the Swedish research council (VR, Grant ID: 2020-01316), and the Swedish governmental agency for innovation systems (VINNOVA, Grant ID: 2017-02960). This research was also funded by the Systems Biology Research Centre at the University of Skövde under grants from the Knowledge Foundation (Grant ID: 20160330).

Lipid nanoparticles (LNPs) are currently used to transport functional mRNAs, such as COVID-19 mRNA vaccines. The delivery of angiogenic molecules, such as therapeutic VEGF-A mRNA, to ischemic tissues for producing new blood vessels is an emerging strategy for the treatment of cardiovascular diseases. Here, the authors deliver VEGF-A mRNA via LNPs and study stoichiometric quantification of their uptake kinetics and how the transport of exogenous LNP-mRNAs between cells is functionally extended by cells’ own vehicles called extracellular vesicles (EVs). The results show that cellular uptake of LNPs and their mRNA molecules occurs quickly, and that the translation of exogenously delivered mRNA begins immediately. Following the VEGF-A mRNA delivery to cells via LNPs, a fraction of internalized VEGF-A mRNA is secreted via EVs. The overexpressed VEGF-A mRNA is detected in EVs secreted from three different cell types. Additionally, RNA-Seq analysis reveals that as cells’ response to LNP-VEGF-A mRNA treatment, several overexpressed proangiogenic transcripts are packaged into EVs. EVs are further deployed to deliver VEGF-A mRNA in vitro and in vivo. Upon equal amount of VEGF-A mRNA delivery via three EV types or LNPs in vitro, EVs from cardiac progenitor cells are the most efficient in promoting angiogenesis per amount of VEGF-A protein produced. Intravenous administration of luciferase mRNA shows that EVs could distribute translatable mRNA to different organs with the highest amounts of luciferase detected in the liver. Direct injections of VEGF-A mRNA (via EVs or LNPs) into mice heart result in locally produced VEGF-A protein without spillover to liver and circulation. In addition, EVs from cardiac progenitor cells cause minimal production of inflammatory cytokines in cardiac tissue compared with all other treatment types. Collectively, the data demonstrate that LNPs transform EVs as functional extensions to distribute therapeutic mRNA between cells, where EVs deliver this, CC BY 4.0© 2023 The Authors. Advanced Science published by Wiley-VCH GmbH.E-mail: hadi.valadi@gu.seThe authors acknowledge the support from the National Genomics Infrastructure in Stockholm funded by Science for Life (SciLife) Laboratory, the Knut and Alice Wallenberg Foundation and the Swedish Research Council, and SNIC/Uppsala Multidisciplinary Center for Advanced Computational Science for assistance with massively parallel sequencing and access to the UPPMAX computational infrastructure. Moreover, the authors acknowledge Mr. Mario Soriano Navarro at the Responsable Servicio Microscopía Electrónica, Valencia Spain, for technical assistance. This work was supported by grants from the Swedish Foundation of Strategic Research (Stiftelsen för strategisk forskning: SSF) in the Industrial Research Centre, FoRmulaEx - Nucleotide Functional Drug Delivery (Grant ID: IRC15-0065), the Swedish research council (VR, Grant ID: 2020-01316), and the Swedish governmental agency for innovation systems (VINNOVA, Grant ID: 2017-02960). This research was also funded by the Systems Biology Research Centre at the University of Skövde under grants from the Knowledge Foundation (Grant ID: 20160330).

Lipid nanoparticles (LNPs) are currently used to transport functional mRNAs, such as COVID-19 mRNA vaccines. The delivery of angiogenic molecules, such as therapeutic VEGF-A mRNA, to ischemic tissues for producing new blood vessels is an emerging strategy for the treatment of cardiovascular diseases. Here, the authors deliver VEGF-A mRNA via LNPs and study stoichiometric quantification of their uptake kinetics and how the transport of exogenous LNP-mRNAs between cells is functionally extended by cells’ own vehicles called extracellular vesicles (EVs). The results show that cellular uptake of LNPs and their mRNA molecules occurs quickly, and that the translation of exogenously delivered mRNA begins immediately. Following the VEGF-A mRNA delivery to cells via LNPs, a fraction of internalized VEGF-A mRNA is secreted via EVs. The overexpressed VEGF-A mRNA is detected in EVs secreted from three different cell types. Additionally, RNA-Seq analysis reveals that as cells’ response to LNP-VEGF-A mRNA treatment, several overexpressed proangiogenic transcripts are packaged into EVs. EVs are further deployed to deliver VEGF-A mRNA in vitro and in vivo. Upon equal amount of VEGF-A mRNA delivery via three EV types or LNPs in vitro, EVs from cardiac progenitor cells are the most efficient in promoting angiogenesis per amount of VEGF-A protein produced. Intravenous administration of luciferase mRNA shows that EVs could distribute translatable mRNA to different organs with the highest amounts of luciferase detected in the liver. Direct injections of VEGF-A mRNA (via EVs or LNPs) into mice heart result in locally produced VEGF-A protein without spillover to liver and circulation. In addition, EVs from cardiac progenitor cells cause minimal production of inflammatory cytokines in cardiac tissue compared with all other treatment types. Collectively, the data demonstrate that LNPs transform EVs as functional extensions to distribute therapeutic mRNA between cells, where EVs deliver this, CC BY 4.0© 2023 The Authors. Advanced Science published by Wiley-VCH GmbH.E-mail: hadi.valadi@gu.seThe authors acknowledge the support from the National Genomics Infrastructure in Stockholm funded by Science for Life (SciLife) Laboratory, the Knut and Alice Wallenberg Foundation and the Swedish Research Council, and SNIC/Uppsala Multidisciplinary Center for Advanced Computational Science for assistance with massively parallel sequencing and access to the UPPMAX computational infrastructure. Moreover, the authors acknowledge Mr. Mario Soriano Navarro at the Responsable Servicio Microscopía Electrónica, Valencia Spain, for technical assistance. This work was supported by grants from the Swedish Foundation of Strategic Research (Stiftelsen för strategisk forskning: SSF) in the Industrial Research Centre, FoRmulaEx - Nucleotide Functional Drug Delivery (Grant ID: IRC15-0065), the Swedish research council (VR, Grant ID: 2020-01316), and the Swedish governmental agency for innovation systems (VINNOVA, Grant ID: 2017-02960). This research was also funded by the Systems Biology Research Centre at the University of Skövde under grants from the Knowledge Foundation (Grant ID: 20160330).

Emerging therapeutic treatments based on the production of proteins by delivering mRNA have become increasingly important in recent times. While lipid nanoparticles (LNPs) are approved vehicles for small interfering RNA delivery, there are still challenges to use this formulation for mRNA delivery. LNPs are typically a mixture of a cationic lipid, distearoylphosphatidylcholine (DSPC), cholesterol, and a PEG-lipid. The structural characterization of mRNA-containing LNPs (mRNA-LNPs) is crucial for a full understanding of the way in which they function, but this information alone is not enough to predict their fate upon entering the bloodstream. The biodistribution and cellular uptake of LNPs are affected by their surface composition as well as by the extracellular proteins present at the site of LNP administration, e.g., apolipoproteinE (ApoE). ApoE, being responsible for fat transport in the body, plays a key role in the LNP’s plasma circulation time. In this work, we use small-angle neutron scattering, together with selective lipid, cholesterol, and solvent deuteration, to elucidate the structure of the LNP and the distribution of the lipid components in the absence and the presence of ApoE. While DSPC and cholesterol are found to be enriched at the surface of the LNPs in buffer, binding of ApoE induces a redistribution of the lipids at the shell and the core, which also impacts the LNP internal structure, causing release of mRNA. The rearrangement of LNP components upon ApoE incubation is discussed in terms of potential relevance to LNP endosomal escape.

Emerging therapeutic treatments based on the production of proteins by delivering mRNA have become increasingly important in recent times. While lipid nanoparticles (LNPs) are approved vehicles for small interfering RNA delivery, there are still challenges to use this formulation for mRNA delivery. LNPs are typically a mixture of a cationic lipid, distearoylphosphatidylcholine (DSPC), cholesterol, and a PEG-lipid. The structural characterization of mRNA-containing LNPs (mRNA-LNPs) is crucial for a full understanding of the way in which they function, but this information alone is not enough to predict their fate upon entering the bloodstream. The biodistribution and cellular uptake of LNPs are affected by their surface composition as well as by the extracellular proteins present at the site of LNP administration, e.g., apolipoproteinE (ApoE). ApoE, being responsible for fat transport in the body, plays a key role in the LNP’s plasma circulation time. In this work, we use small-angle neutron scattering, together with selective lipid, cholesterol, and solvent deuteration, to elucidate the structure of the LNP and the distribution of the lipid components in the absence and the presence of ApoE. While DSPC and cholesterol are found to be enriched at the surface of the LNPs in buffer, binding of ApoE induces a redistribution of the lipids at the shell and the core, which also impacts the LNP internal structure, causing release of mRNA. The rearrangement of LNP components upon ApoE incubation is discussed in terms of potential relevance to LNP endosomal escape.

Lipid nanoparticles (LNPs) are promising drug and gene carriers. Upon intravenous administration, LNPs' experience different degree of cellular uptake depending on their formulation. Currently, in vitro and in vivo studies are the gold standard for assessing the fate of nano carriers once administered, but they are time consuming and expensive. In this work, we propose a time and cost-effective method to screen a wide range of LNP formulations and select the most promising candidates for in vitro and in vivo studies. Two different approaches were explored to investigate the binding affinity between LNPs and serum proteins using sensor functionalisation with either protein specific antibody or PEG specific antibody. The first approach allowed to identify the presence of a specific protein in the protein corona of lipid particles (reconstituted and native high-density lipoproteins (rHDL and HDL), and low-density lipoproteins LDL); while the second one provided a versatile platform for the immobilisation of pegylated-particles in order to follow the interaction with serum proteins and hence predict the composition of LNP protein corona. Sensing was done using Quartz Crystal Microbalance with Dissipation (QCM-D) but the approach is extendable to other surface sensing techniques such as Surface Plasmon Resonance (SPR) or ellipsometry.

Lipid nanoparticles (LNPs) are promising drug and gene carriers. Upon intravenous administration, LNPs' experience different degree of cellular uptake depending on their formulation. Currently, in vitro and in vivo studies are the gold standard for assessing the fate of nano carriers once administered, but they are time consuming and expensive. In this work, we propose a time and cost-effective method to screen a wide range of LNP formulations and select the most promising candidates for in vitro and in vivo studies. Two different approaches were explored to investigate the binding affinity between LNPs and serum proteins using sensor functionalisation with either protein specific antibody or PEG specific antibody. The first approach allowed to identify the presence of a specific protein in the protein corona of lipid particles (reconstituted and native high-density lipoproteins (rHDL and HDL), and low-density lipoproteins LDL); while the second one provided a versatile platform for the immobilisation of pegylated-particles in order to follow the interaction with serum proteins and hence predict the composition of LNP protein corona. Sensing was done using Quartz Crystal Microbalance with Dissipation (QCM-D) but the approach is extendable to other surface sensing techniques such as Surface Plasmon Resonance (SPR) or ellipsometry.

Emerging therapeutic treatments based on the production of proteins by delivering mRNA have become increasingly important in recent times. While lipid nanoparticles (LNPs) are approved vehicles for small interfering RNA delivery, there are still challenges to use this formulation for mRNA delivery. LNPs are typically a mixture of a cationic lipid, distearoylphosphatidylcholine (DSPC), cholesterol, and a PEG-lipid. The structural characterization of mRNA-containing LNPs (mRNA-LNPs) is crucial for a full understanding of the way in which they function, but this information alone is not enough to predict their fate upon entering the bloodstream. The biodistribution and cellular uptake of LNPs are affected by their surface composition as well as by the extracellular proteins present at the site of LNP administration, e.g., apolipoproteinE (ApoE). ApoE, being responsible for fat transport in the body, plays a key role in the LNP’s plasma circulation time. In this work, we use small-angle neutron scattering, together with selective lipid, cholesterol, and solvent deuteration, to elucidate the structure of the LNP and the distribution of the lipid components in the absence and the presence of ApoE. While DSPC and cholesterol are found to be enriched at the surface of the LNPs in buffer, binding of ApoE induces a redistribution of the lipids at the shell and the core, which also impacts the LNP internal structure, causing release of mRNA. The rearrangement of LNP components upon ApoE incubation is discussed in terms of potential relevance to LNP endosomal escape.

Emerging therapeutic treatments based on the production of proteins by delivering mRNA have become increasingly important in recent times. While lipid nanoparticles (LNPs) are approved vehicles for small interfering RNA delivery, there are still challenges to use this formulation for mRNA delivery. LNPs are typically a mixture of a cationic lipid, distearoylphosphatidylcholine (DSPC), cholesterol, and a PEG-lipid. The structural characterization of mRNA-containing LNPs (mRNA-LNPs) is crucial for a full understanding of the way in which they function, but this information alone is not enough to predict their fate upon entering the bloodstream. The biodistribution and cellular uptake of LNPs are affected by their surface composition as well as by the extracellular proteins present at the site of LNP administration, e.g., apolipoproteinE (ApoE). ApoE, being responsible for fat transport in the body, plays a key role in the LNP’s plasma circulation time. In this work, we use small-angle neutron scattering, together with selective lipid, cholesterol, and solvent deuteration, to elucidate the structure of the LNP and the distribution of the lipid components in the absence and the presence of ApoE. While DSPC and cholesterol are found to be enriched at the surface of the LNPs in buffer, binding of ApoE induces a redistribution of the lipids at the shell and the core, which also impacts the LNP internal structure, causing release of mRNA. The rearrangement of LNP components upon ApoE incubation is discussed in terms of potential relevance to LNP endosomal escape.

Secondary nucleation, wherein crystal seeds are used to induce crystallization, is widely employed in industrial crystallizations. Despite its significance, our understanding of the process, particularly at the molecular level, remains rudimentary. An outstanding question is why do a few seeds give rise to a many-fold increase in new crystals? Using molecular simulation coupled with experiments we have uncovered the molecular processes that give rise to this autocatalytic behavior. The simulations reveal formation of molecular aggregates in solution, which on coming in contact with the surface of the seed undergo nucleation to form new crystallites. These crystallites are weakly bound to the crystal surface and can be readily sheared by fluid, making the seed surfaces available again to repeat the process. Further, the new crystallites on development can in turn serve as seeds. This mechanistic insight will enable better control in engineering crystalline products to design.

Secondary nucleation, wherein crystal seeds are used to induce crystallization, is widely employed in industrial crystallizations. Despite its significance, our understanding of the process, particularly at the molecular level, remains rudimentary. An outstanding question is why do a few seeds give rise to a many-fold increase in new crystals? Using molecular simulation coupled with experiments we have uncovered the molecular processes that give rise to this autocatalytic behavior. The simulations reveal formation of molecular aggregates in solution, which on coming in contact with the surface of the seed undergo nucleation to form new crystallites. These crystallites are weakly bound to the crystal surface and can be readily sheared by fluid, making the seed surfaces available again to repeat the process. Further, the new crystallites on development can in turn serve as seeds. This mechanistic insight will enable better control in engineering crystalline products to design.

Oral drug delivery is the predominant administration route for a major part of the pharmaceutical products used worldwide. Further understanding and improvement of gastrointestinal drug absorption predictions is currently a highly prioritized area of research within the pharmaceutical industry. The fraction absorbed (f(abs)) of an oral dose after administration of a solid dosage form is a key parameter in the estimation of the in vivo performance of an orally administrated drug formulation. This study discloses an evaluation of the predictive performance of the mechanistic physiologically based absorption model GI-Sim. GI-Sim deploys a compartmental gastrointestinal absorption and transit model as well as algorithms describing permeability, dissolution rate, salt effects, partitioning into micelles, particle and micelle drifting in the aqueous boundary layer, particle growth and amorphous or crystalline precipitation. Twelve APIs with reported or expected absorption limitations in humans, due to permeability, dissolution and/or solubility, were investigated. Predictions of the intestinal absorption for different doses and formulations were performed based on physicochemical and biopharmaceutical properties, such as solubility in buffer and simulated intestinal fluid, molecular weight, pK(a), diffusivity and molecule density, measured or estimated human effective permeability and particle size distribution. The performance of GI-Sim was evaluated by comparing predicted plasma concentration time profiles along with oral pharmacokinetic parameters originating from clinical studies in healthy individuals. The capability of GI-Sim to correctly predict impact of dose and particle size as well as the in vivo performance of nanoformulations was also investigated. The overall predictive performance of GI-Sim was good as >95% of the predicted pharmacokinetic parameters (C-max and AUC) were within a 2-fold deviation from the clinical observations and the predicted plasma AU

The purpose of this study was to investigate in viva intestinal precipitation of a model drug mebendazole, a basic BCS class II drug, using dogs with intestinal stomas for administration or sampling. After oral administration of a solution with an expected intestinal supersaturation of approximately 20 times the solubility, the measured supersaturation in dog intestinal fluid (DIE) was up to 10 times and, on average, only 11% of the given dose was retrieved as solid drug in the collected fluid from the stoma. The drug was rapidly absorbed with >90% of the total systemic exposure reached within three hours after duodenal administration of a solution. In silico absorption modeling showed that in vivo data were reasonably well described by a nonprecipitating solution. An in vitro model of precipitation in DIF predicted that the intestinal concentration of dissolved mebendazole would be less than 1/5 of the initial concentration within 10 min at concentrations comparable to in vivo. It was concluded that intestinal precipitation did not have any major influence on mebendazole absorption. The extent of precipitation was overpredicted in vitro given the in vivo absorption rate, and further work is needed to identify in vitro factors that could enable more accurate in vivo predictions of intestinal precipitation from solutions.

PURPOSE: To investigate the prediction accuracy of in vitro and in vitro/in silico methods for in vivo intestinal precipitation of basic BCS class II drugs in humans. METHODS: Precipitation rate of a model drug substance, AZD0865 (pKa = 6.1; log K(D) = 4.2), was investigated in vitro using simulated intestinal media, and calculations of the crystallization rates were made with a theoretical model. Human intestinal precipitation was estimated by analysis of pharmacokinetic data from clinical studies at different doses. RESULTS: All in vitro models predicted rapid drug precipitation, where the intestinal concentration of dissolved AZD0865 at the highest dose tested was expected to decrease to half after less than 20 min. However, there was no indication of precipitation in vivo in humans as there was a dose proportional increase in drug plasma exposure. The theoretical model predicted no significant precipitation within the range of expected in vivo intestinal concentrations. CONCLUSIONS: This study indicated that simple in vitro methods of in vivo precipitation of orally administered bases overpredict the intestinal crystalline precipitation in vivo in humans. Hydrodynamic conditions were identified as one important factor that needs to be better addressed in future in vivo predictive methods.

The crystallization process in aqueous solutions of the drug bicalutamide and the effect of the polymer polyvinylpyrrolidone (PVP) have been studied. Results show that PVP decreased the crystallization rate significantly in a system with PVP concentrations as low as 0.01% (w/w), without changing the polymorph formed. The crystal habit was changed already at PVP concentrations as low as 0.001% (w/w). Measurements made with self-diffusion NMR indicated that the decrease in crystallization rate was not because of a reduced supersaturation due to bicalutamide binding to PVP in solution. Furthermore, in experiments designed to specifically study crystal nucleation, the same nucleation rate was found in the absence and presence of PVP. Instead, PVP adsorbes to the crystals formed in solution and by doing so, the crystal growth rate is reduced. This was confirmed in separate experiments using bicalutamide nanocrystals. By combining theories describing classical nucleation and crystal growth, with some modifications, a consistent description of several independent experiments performed in polymer-free systems was obtained. From these experiments a crystal-water interfacial tension of 22.1 mN/m was extracted. We also analyze the interfacial tension of other crystalline organic solids and find that it varies approximately as the logarithm of the solubility. This finding is discussed within the framework of the Bragg-Williams regular solution theory where we also compare with the tension of liquid alkanes.

The aim of this work was to evaluate an in vitro-in silico approach for prediction of small intestinal crystallization of two weakly basic model BCS class II drugs, AZD0865 and mebendazole, and the effect crystallization would have on the absorption prediction of the drug. The crystallization rates were investigated in an in vitro method using simulated gastric and intestinal media, and the result was modeled by using Classical Nucleation Theory (CNT). The effect of varying in vitro parameters (initial drug concentration, rate of mixing gastric and intestinal fluid, stirring and filtration) on the interfacial tension g, being a key parameter in CNT, was investigated. The initial drug concentration had the most significant effect on g for both substances tested, although g is a fundamental parameter independent of concentration according to CNT. In the subsequent in silico prediction of drug absorption an empirical approach was used where g was predicted at expected in vivo small intestinal concentrations. The results showed that lack of crystallization effects on absorption in man of the model drug AZD0865 up to doses of 4 mg/kg could be predicted. Mebendazole intestinal precipitation in canines was also well described by the model, where mean predicted amount precipitated was 111% (range 41-166%) of measured solid amount, and mean predicted supersaturation was 106% (range 73-118%) of measured supersaturation. The plasma concentration of mebendazole after duodenal administration of a solution could not be predicted by the model with the same precision in the absence of measured intestinal drug concentrations as basis for estimating the g value. In conclusion, the in vitro-in silico approach can be used for predictions of absorption effects of crystallization, but the model could benefit from further development work on the theoretical crystallization model and in vitro experimental design.