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

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

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.

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

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.

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

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 in vitro studies are promising, following in vivo 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 vivo. 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 in vivo. 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 in vivo studies.

Efficient Delivery of DNA Using Lipid Nanoparticles.

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.

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

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.

A review of lifestyle and environment risk factors for pancreatic cancer.

Pancreatic cancer (PaCa) is one of the deadliest cancers known and its incidence is increasing in the developed countries. Because of the lack of biomarkers that allow early detection and the tendency of the disease to be asymptomatic, the diagnosis comes often too late for effective surgical or chemotherapy intervention. Lifestyle factors, that may cause common genetic modifications occurring in the disease, interfere with pancreatic physiology or function, and play a role in PaCa development, have been of concern recently, since a strategy to prevent this severe cancer is needed. This review identifies the latest evidences related to increased risk of developing PaCa due to dietary habits such as high alcohol, fructose and red or processed meat intake, and pathological conditions such as diabetes, obesity and infections in addition to stress and smoking behaviour. It aims to highlight the importance of intervening on modifiable risk factors: the action on these factors could prevent a considerable number of new cases of PaCa.

mTOR Pathway in Gastroenteropancreatic Neuroendocrine Tumor (GEP-NETs).

Gastroenteropancreatic neuroendocrine neoplasms (GEP-NENs) originate from neuroendocrine cells in the gastrointestinal tract. They are heterogeneous, and though initially considered rare tumors, the incidence of GEP-NENs has increased in the last few decades. Therapeutic approaches for the metastatic disease include surgery, radiological intervention by chemoembolisation, radiofrequency ablation, biological therapy in addition to somatostatin analogs, and PRRT therapy (177Lu-DOTATATE). The PI3K-AKT-mTOR pathway is essential in the regulation of protein translation, cell growth, and metabolism. Evidence suggests that the mTOR pathway is involved in malignant progression and resistance to treatment through over-activation of several mechanisms. PI3K, one of the main downstream of the Akt-mTOR axis, is mainly involved in the neoplastic process. This pathway is frequently deregulated in human tumors, making it a central target in the development of new anti-cancer treatments. Recent molecular studies identify potential targets within the PI3K/Akt/mTOR pathway in GEP-NENs. However, the use of target therapy has been known to lead to resistance due to several mechanisms such as feedback activation of alternative pathways, inactivation of protein kinases, and deregulation of the downstream mTOR components. Therefore, the specific role of targeted drugs for the management of GEP-NENs is yet to be well-defined. The variable clinical presentation of advanced neuroendocrine tumors is a significant challenge for designing studies. This review aims to highlight the role of the PI3K/Akt/mTOR pathway in the development of neuroendocrine tumors and further specify its potential as a therapeutic target in advanced stages.