standardized-sds-page-workflow-for-personalized-protein-corona

Standardized SDS-PAGE Workflow for Personalized Protein Corona Profiling in Early Cancer Detection

The formation of a personalized protein corona (PC) on nanoparticles (NPs) upon exposure to biological fluids has emerged as a promising, minimally ...

Sapienza University of Rome

In recent years, lipid nanoparticles (LNPs) have gained considerable attention in numerous research fields ranging from gene therapy to cancer immunotherapy and DNA vaccination. While some RNA-encapsulating LNP formulations passed clinical trials, DNA-loaded LNPs have been only marginally explored so far. To fulfil this gap, herein we investigated the effect of several factors influencing the microfluidic formulation and transfection behavior of DNA-loaded LNPs such as PEGylation, total flow rate (TFR), concentration and particle density at the cell surface. We show that PEGylation and post-synthesis sample concentration facilitated formulation of homogeneous and small size LNPs with high transfection efficiency and minor, if any, cytotoxicity on human Embryonic Kidney293 (HEK-293), spontaneously immortalized human keratinocytes (HaCaT), immortalized keratinocytes (N/TERT) generated from the transduction of human primary keratinocytes, and epidermoid cervical cancer (CaSki) cell lines. On the other side, increasing TFR had a detrimental effect both on the physicochemical properties and transfection properties of LNPs. Lastly, the effect of particle concentration at the cell surface on the transfection efficiency (TE) and cell viability was largely dependent on the cell line, suggesting that its case-by-case optimization would be necessary. Overall, we demonstrate that fine tuning formulation and microfluidic parameters is a vital step for the generation of highly efficient DNA-loaded LNPs.

Microfluidic Formulation of DNA-Loaded Multicomponent Lipid Nanoparticles for Gene Delivery.

DNA vaccination has been extensively studied as a promising strategy for tumor treatment. Despite the efforts, the therapeutic efficacy of DNA vaccines has been limited by their intrinsic poor cellular internalization. Electroporation, which is based on the application of a controlled electric field to enhance DNA penetration into cells, has been the method of choice to produce acceptable levels of gene transfer in vivo. However, this method may cause cell damage or rupture, non-specific targeting, and even degradation of pDNA. Skin irritation, muscle contractions, pain, alterations in skin structure, and irreversible cell damage have been frequently reported. To overcome these limitations, in this work, we use a microfluidic platform to generate DNA-loaded lipid nanoparticles (LNPs) which are then characterized by a combination of dynamic light scattering (DLS), synchrotron small-angle X-ray scattering (SAXS), and transmission electron microscopy (TEM). Despite the clinical successes obtained by LNPs for mRNA and siRNA delivery, little is known about LNPs encapsulating bulkier DNA molecules, the clinical application of which remains challenging. For in vitro screening, LNPs were administered to human embryonic kidney 293 (HEK-293) and Chinese hamster ovary (CHO) cell lines and ranked for their transfection efficiency (TE) and cytotoxicity. The LNP formulation exhibiting the highest TE and the lowest cytotoxicity was then tested for the delivery of the DNA vaccine pVAX-hECTM targeting the human neoantigen HER2, an oncoprotein overexpressed in several cancer types. Using fluorescence-activated cell sorting (FACS), immunofluorescence assays and fluorescence confocal microscopy (FCS), we proved that pVAX-hECTM-loaded LNPs produce massive expression of the HER2 antigen on the cell membrane of HEK-293 cells. Our results provide new insights into the structure-activity relationship of DNA-loaded LNPs and pave the way for the access of this gene delivery technology to preclinical studies.

Efficient Delivery of DNA Using Lipid Nanoparticles.

In the last decade, graphene oxide (GO)-based nanomaterials have attracted much attention for their potential anti-cancer properties against various cancer cell types. However, while  studies are promising, following  investigations fail to show any relevant efficacy. Recent research has clarified that the wide gap between benchtop discoveries and clinical practice is due to our limited knowledge about the physical-chemical transformation of nanomaterials . In physiological environments, nanomaterials are quickly coated by a complex dress of biological molecules referred to as the protein corona. Mediating the interaction between the pristine material and the biological system the protein corona controls the mechanisms of action of nanomaterials up to the sub-cellular level. Here we investigate the anticancer ability of GO in SK-BR-3 human breast cancer cells over-expressing the human epidermal growth factor receptor 2 (HER-2), which is functionally implicated in the cell growth and proliferation through the activation of downstream pathways, including the PI3K/AKT/mTOR and MAPK/ERK signaling cascades. Western blot analysis demonstrated that GO treatment resulted in a marked decrease in total HER-2, associated with a down-regulation of the expression and activation of protein kinase B (AKT) and extracellular signal-regulated kinase (ERK) thus indicating that GO may act as a potent HER-2 inhibitor. On the other side, the protein corona reverted the effects of GO on HER-2 expression and molecular downstream events to the control level. Our findings may suggest a mechanistic explanation of the reduced anticancer properties of GO-based nanomaterials  These results may also represent a good prediction strategy for the anticancer activity of nanomaterials designed for biomedical purposes, reaffirming the necessity of exploring their effectiveness under physiologically relevant conditions before moving on to the next  studies.

The protein corona reduces the anticancer effect of graphene oxide in HER-2-positive cancer cells.

Lipid nanoparticles (LNPs) are currently having an increasing impact on nanomedicines as delivery agents, among others, of RNA molecules (e.g., short interfering RNA for the treatment of hereditary diseases or messenger RNA for the development of COVID-19 vaccines). Despite this, the delivery of plasmid DNA (pDNA) by LNPs in preclinical studies is still unsatisfactory, mainly due to the lack of systematic structural and functional studies on DNA-loaded LNPs. To tackle this issue, we developed, characterized, and tested a library of 16 multicomponent DNA-loaded LNPs which were prepared by microfluidics and differed in lipid composition, surface functionalization, and manufacturing factors. 8 out of 16 formulations exhibited proper size and zeta potential and passed to the validation step, that is, the simultaneous quantification of transfection efficiency and cell viability in human embryonic kidney cells (HEK-293). The most efficient formulation (LNP15) was then successfully validated both in vitro, in an immortalized adult keratinocyte cell line (HaCaT) and in an epidermoid cervical cancer cell line (CaSki), and in vivo as a nanocarrier to deliver a cancer vaccine against the benchmark target tyrosine-kinase receptor HER2 in C57BL/6 mice. Finally, by a combination of confocal microscopy, transmission electron microscopy and synchrotron small-angle X-ray scattering, we were able to show that the superior efficiency of LNP15 can be linked to its disordered nanostructure consisting of small-size unoriented layers of pDNA sandwiched between closely apposed lipid membranes that undergo massive destabilization upon interaction with cellular lipids. Our results provide new insights into the structure-activity relationship of pDNA-loaded LNPs and pave the way to the clinical translation of this gene delivery technology.

Mechanistic Insights into the Superior DNA Delivery Efficiency of Multicomponent Lipid Nanoparticles: An In Vitro and In Vivo Study.

PEGylated lipid nanoparticles (LNPs) are commonly used to deliver bioactive molecules, but the role of PEGylation in DNA-loaded LNP interactions at the cellular and subcellular levels remains poorly understood. In this study, we investigated the mechanism of action of DNA-loaded PEGylated LNPs using gene reporter technologies, dynamic light scattering (DLS), synchrotron small angle X-ray scattering (SAXS), and fluorescence confocal microscopy (FCS). We found that PEG has no significant impact on the size or nanostructure of DNA LNPs but reduces their zeta potential and interaction with anionic cell membranes. PEGylation increases the structural stability of LNPs and results in lower DNA unloading. FCS experiments revealed that PEGylated LNPs are internalized intact inside cells and largely shuttled to lysosomes, while unPEGylated LNPs undergo massive destabilization on the plasma membrane. These findings can inform the design, optimization, and validation of DNA-loaded LNPs for gene delivery and vaccine development.

Investigating the mechanism of action of DNA-loaded PEGylated lipid nanoparticles.

The existing manufacturing protocols for CAR-T cell therapies pose notable challenges, particularly in attaining a transient transfection that endures for a significant duration. To address this gap, this study aims to formulate a transfection protocol utilizing multiple lipid-based nanoparticles (LNPs) administrations to enhance transfection efficiency (TE) to clinically relevant levels. By systematically fine-tuning and optimizing our transfection protocol through a series of iterative refinements, we have accomplished a remarkable one-order-of-magnitude augmentation in TE within the immortalized T-lymphocyte Jurkat cell line. This enhancement has been consistently observed over 2 weeks, and importantly, it has been achieved without any detrimental impact on cell viability. In the subsequent phase of our study, we aimed to optimize the gene delivery system by evaluating three lipid-based formulations tailored for DNA encapsulation using our refined protocol. These formulations encompassed two LNPs constructed from ionizable lipids and featuring systematic variations in lipid composition (iLNPs) and a cationic lipoplex (cLNP). Our findings showcased a notable standout among the three formulations, with cLNP emerging as a frontrunner for further refinement and integration into the production pipeline of CAR-T therapies. Consequently, cLNP was scrutinized for its potential to deliver CAR-encoding plasmid DNA to the HEK-293 cell line. Confocal microscopy experiments demonstrated its efficiency, revealing substantial internalization compared to iLNPs. By employing a recently developed confocal image analysis method, we substantiated that cellular entry of cLNP predominantly occurs through macropinocytosis. This mechanism leads to heightened intracellular endosomal escape and mitigates lysosomal accumulation. The successful expression of anti-CD19-CD28-CD3z, a CAR engineered to target CD19, a protein often expressed on the surface of B cells, was confirmed using a fluorescence-based assay. Overall, our results indicated the effectiveness of cLNP in gene delivery and suggested the potential of multiple administration transfection as a practical approach for refining T-cell engineering protocols in CAR therapies. Future investigations may focus on refining outcomes by adjusting transfection parameters like nucleic acid concentration, lipid-to-DNA ratio, and incubation time to achieve improved TE and increased gene expression levels.

Optimizing Transfection Efficiency in CAR-T Cell Manufacturing through Multiple Administrations of Lipid-Based Nanoparticles.

Currently available vaccines against COVID-19 showed high efficacy against the original strain of SARS-CoV-2 but progressively lower efficacy against new variants. In response to emerging SARS-CoV-2 strains, we propose chimeric DNA vaccines encoding the spike antigen, including a combination of selected key mutations from different variants of concern. We developed two DNA vaccines, pVAX-S1-TM-D614G and pVAX-S1-TM-INDUK (INDUK), encoding the SARS-CoV-2 S1 spike subunit in fusion with the transmembrane region that allows protein trimerization as predicted by  analysis. pVAX-S1-TM-D614G included the dominant D614G substitution, while the chimeric vaccine INDUK contained additional selected mutations from the Delta (E484Q and L452R) and Alpha (N501Y and A570D) variants. Considering that aging is a risk factor for severe disease and that suboptimal vaccine responses were observed in older individuals, the immunogenicity of pVAX-S1-TM-D614G and INDUK was tested in both young and aged C57BL/6 mice. Two vaccine doses were able to trigger significant anti-SARS-CoV-2 antibody production, showing neutralizing activity. ELISA tests confirmed that antibodies induced by pVAX-S1-TM-D614G and INDUK were able to recognize both Wuhan Spike and Delta variant Spike as trimers, while neutralizing antibodies were detected by an ACE2:SARS-CoV-2 Spike S1 inhibitor screening assay, designed to assess the capacity of antibodies to block the interaction between the viral spike S1 protein and the ACE2 receptor. Although antibody titer declined within six months, a third booster dose significantly increased the magnitude of humoral response, even in aged individuals, suggesting that immune recall can improve antibody response durability. The analysis of cellular responses demonstrated that vaccination with INDUK elicited an increase in the percentage of SARS-CoV-2-specific IFN-γ producing T lymphocytes in immunized young mice and TNF-α-producing T lymphocytes in both young and aged mice. These findings not only hold immediate promise for addressing evolving challenges in SARS-CoV-2 vaccination but also open avenues to refine strategies and elevate the effectiveness of next-generation vaccines.

Enhancing Immune Responses against SARS-CoV-2 Variants in Aged Mice with INDUK: A Chimeric DNA Vaccine Encoding the Spike S1-TM Subunits.

Polyethylene glycol (PEG) is a common surface modification for lipid nanoparticles (LNPs) to improve their stability and in vivo circulation time. However, the impact of PEGylation on LNP cellular uptake remains poorly understood. To tackle this issue, we systematically compared plain and PEGylated LNPs by combining dynamic light scattering, electrophoretic light scattering, and synchrotron small-angle X-ray scattering (SAXS) that unveils a striking similarity in size and core structure but a significant reduction in surface charge. Upon administration to human embryonic kidney (HEK 293) cells, plain and PEGylated LNPs were internalized through different endocytic routes, as revealed by spatiotemporal correlation spectroscopy. An imaging-derived mean square displacement (iMSD) analysis shows that PEGylated LNPs exhibit a significantly stronger preference for caveolae-mediated endocytosis (CAV) and clathrin-mediated endocytosis (CME) pathways compared to plain LNPs, with these latter being better tailored to MCR-dependent internalization and trafficking. This suggests that PEG plays a crucial role in directing LNPs toward specific cellular uptake routes. Further studies should explore how PEG-mediated endocytosis impacts intracellular trafficking and ultimately translates to therapeutic efficacy, guiding the design of next-generation LNP delivery systems.

PEGylation-Dependent Cell Uptake of Lipid Nanoparticles Revealed by Spatiotemporal Correlation Spectroscopy.

Lipid nanoparticles (LNPs) play a crucial role in addressing genetic disorders, and cancer, and combating pandemics such as COVID-19 and its variants. Yet, the ability of LNPs to effectively encapsulate large-size DNA molecules remains elusive. This is a significant limitation, as the successful delivery of large-size DNA holds immense potential for gene therapy. To address this gap, the present study focuses on the design of PEGylated LNPs, incorporating large-sized DNA, departing from traditional RNA and ionizable lipids. The resultant LNPs demonstrate a unique particle morphology. These particles were further engineered with a DNA coating and plasma proteins. This multicomponent bionanoconstruct exhibits enhanced transfection efficiency and safety in controlled laboratory settings and improved immune system evasion in in vivo tests. These findings provide valuable insights for the design and development of bionanoarchitectures for large-size DNA delivery, opening new avenues for transformative gene therapies.

Structuring lipid nanoparticles, DNA, and protein corona into stealth bionanoarchitectures for in vivo gene delivery.

Among the various types of pancreatic cancers, pancreatic ductal adenocarcinoma (PDAC) is the most lethal and aggressive, due to its tendency to metastasize quickly and has a particularly low five-year survival rate. Carbohydrate antigen 19-9 (CA 19-9) is the only biomarker approved by the Food and Drug Administration for PDAC and has been a focal point in diagnostic strategies, but its sensitivity and specificity are not sufficient for early and accurate detection. To address this issue, we introduce a synergistic approach combining CA 19-9 levels with a graphene oxide (GO)-based blood test. This non-invasive technique relies on the analysis of personalized protein corona formed on GO sheets once they are embedded in human plasma. Pairing CA 19-9 values with GO protein patterns from  = 106 donors significantly improved the ability to differentiate between non-oncological and PDAC patients (up to 92%), also boosting the classification of PDAC subjects by 50% compared to CA 19-9 testing alone. Overall, this study sought to bridge the existing gaps in PDAC detection by exploiting the complementary strengths of conventional biomarkers and cutting-edge nanotechnology. Exploration of this combined strategy holds promise for advancing the early detection of PDAC, ultimately contributing to improved patient prognosis and treatment outcomes.

Nanoparticle-protein corona enhances accuracy of Ca-19.9-based pancreatic cancer classification.

The advent of chimeric antigen receptor (CAR) T-cell therapy has introduced a novel and personalized approach to cancer treatment. Despite its promise, the challenge of developing a system that bypasses the need for viral vectors remains significant, particularly in terms of achieving a clinical efficacy and sustained durability. To address these challenges, lipid nanoparticles (LNPs) produced through advanced microfluidic technology have been recently utilized to encapsulate plasmid DNA (pDNA) encoding CAR receptors. However, the intrinsic challenges associated with pDNA encapsulation, along with the critical requirement for efficient expression, remain substantial obstacles. Here, we show that incorporating DNA-condensing agents into the microfluidic manufacturing of LNPs effectively overcomes these limitations. Briefly, we conducted a preliminary investigation to characterize LNPs with and without the commercial condensing agent P3000-Reagent (PR), focusing on their physicochemical properties and scrutinizing the biological outcomes primarily in the HEK-293 cell line. Our results demonstrated that precondensation of the pDNA with PR differentially increased the transfection efficiency of the tested formulations, whereas confocal microscopy indicated reduced lysosomal colocalization and major nuclear localization. Finally, PR was found to enhance LNP efficiency upon multiple administrations to the immortalized T-lymphocyte Jurkat cell line, enabling the delivery of both a luciferase reporter gene and a functional CAR-encoding plasmid. Overall, these findings underscore the great potential of introducing DNA-condensing agents into the LNP preparation process, especially for systems designed for challenging delivery applications, such as multiadministration transfection protocols.

Article	Year
Microfluidic Formulation of DNA-Loaded Multicomponent Lipid Nanoparticles for Gene Delivery. Pharmaceutics\| PubMed ID: 34452253	2021
Efficient Delivery of DNA Using Lipid Nanoparticles. Pharmaceutics\| PubMed ID: 36015328	2022
The protein corona reduces the anticancer effect of graphene oxide in HER-2-positive cancer cells. Nanoscale advances\| PubMed ID: 36133348	2022
Mechanistic Insights into the Superior DNA Delivery Efficiency of Multicomponent Lipid Nanoparticles: An In Vitro and In Vivo Study. ACS applied materials & interfaces\| PubMed ID: 36524967	2022
Investigating the mechanism of action of DNA-loaded PEGylated lipid nanoparticles. Nanomedicine : nanotechnology, biology, and medicine\| PubMed ID: 37507061	2023
Optimizing Transfection Efficiency in CAR-T Cell Manufacturing through Multiple Administrations of Lipid-Based Nanoparticles. ACS applied bio materials\| PubMed ID: 38775109	2024
Enhancing Immune Responses against SARS-CoV-2 Variants in Aged Mice with INDUK: A Chimeric DNA Vaccine Encoding the Spike S1-TM Subunits. ACS omega\| PubMed ID: 39157118	2024
PEGylation-Dependent Cell Uptake of Lipid Nanoparticles Revealed by Spatiotemporal Correlation Spectroscopy. ACS pharmacology & translational science\| PubMed ID: 39421655	2024
Structuring lipid nanoparticles, DNA, and protein corona into stealth bionanoarchitectures for in vivo gene delivery. Nature communications\| PubMed ID: 39438484	2024
Nanoparticle-protein corona enhances accuracy of Ca-19.9-based pancreatic cancer classification. Nanoscale\| PubMed ID: 39868525	2025
Precondensed Plasmid DNA Enhances CAR‑T Cell Generation via Lipid Nanoparticles. ACS omega\| PubMed ID: 40686987	2025

Serena Renzi

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