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Messenger RNA (mRNA) can treat genetic disease using protein replacement or genome editing approaches but requires a suitable carrier to circumnavigate biological barriers and access the desired cell type within the target organ. Lipid nanoparticles (LNPs) are widely used in the clinic for mRNA delivery yet are limited in their applications due to significant hepatic accumulation because of the formation of a protein corona enriched in apolipoprotein E (ApoE). Our lab developed selective organ targeting (SORT) LNPs that incorporate a supplementary component, termed a SORT molecule, for tissue-specific mRNA delivery to the liver, spleen, and lungs of mice. Mechanistic work revealed that the biophysical class of SORT molecule added to the LNP forms a distinct protein corona that helps determine where in the body mRNA is delivered. To better understand which plasma proteins could drive tissue-specific mRNA delivery, we characterized a panel of quaternary ammonium lipids as SORT molecules to assess how chemical structure affects the organ-targeting outcomes and protein corona of lung-targeting SORT LNPs. We discovered that variations in the chemical structure of both the lipid alkyl tail and headgroup impact the potency and specificity of mRNA delivery to the lungs. Furthermore, changes to the chemical structure alter the quantities and identities of protein corona constituents in a manner that correlates with organ-targeting outcomes, with certain proteins appearing to promote lung targeting whereas others reduce delivery to off-target organs. These findings unveil a nuanced relationship between LNP chemistry and endogenous targeting, where the ensemble of proteins associated with an LNP can play various roles in determining the tissue-specificity of mRNA delivery, providing further design criteria for optimization of clinically-relevant nanoparticles for extrahepatic delivery of genetic payloads. [Display omitted] [ABSTRACT FROM AUTHOR]

The delivery of mRNA molecules to organs beyond the liver is valuable for therapeutic applications. Functionalized lipid nanoparticles (LNPs) using exogenous mechanisms can regulate in vivo mRNA expression profiles from hepatocytes to extrahepatic tissues but lead to process complexity and cost escalation. Here, we report that mRNA expression gradually shifts from the liver to the spleen in an ionizable lipid tail length-dependent manner. Remarkably, this simple chemical strategy held true even when different ionizable lipid head structures were employed. As a potential mechanism underlying this discovery, our data suggest that 1,2-distearoyl- sn -glycero-3-phosphocholine (DSPC) is enriched on the surface of mRNA/LNPs with short-tail lipids. This feature limits their interaction with biological components, avoiding their rapid hepatic clearance. We also show that spleen-targeting LNPs loaded with SARS-CoV-2 receptor-binding domain (RBD) mRNA can efficiently induce immune responses and neutralize activity following intramuscular vaccination priming and boosting.

Aya Ahmed Sebak,1 Iman Emam Omar Gomaa,2 Aliaa Nabil ElMeshad,3 Mahmoud Hussien Farag,1 Ulrike Breitinger,4 Hans-Georg Breitinger,4 Mahmoud Hashem AbdelKader5,6 1Pharmaceutical Technology Department, Faculty of Pharmacy and Biotechnology, German University in Cairo (GUC), New Cairo City, Egypt; 2Biochemistry Department, Faculty of Pharmacy, October University for Modern Sciences and Arts (MSA), Giza, Egypt; 3Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, Cairo University, Cairo, Egypt; 4Biochemistry Department, Faculty of Pharmacy and Biotechnology, German University in Cairo (GUC), New Cairo City, Egypt; 5National Institute of Laser Enhanced Sciences (NILES), Cairo University (CU), Giza, Egypt; 6European University in Egypt (EUE), New Administrative Capital, Cairo, EgyptCorrespondence: Aya Ahmed SebakFaculty of Pharmacy and Biotechnology, German University in Cairo (GUC), New Cairo City, EgyptTel +20 1205727787Email aya.sebak@gmail.comIman Emam Omar GomaaBiochemistry Department Faculty of Pharmacy, October University for Modern Sciences and Arts (MSA), Giza, EgyptTel +20 1275146432Email gomaa.iman@gmail.comIntroduction: Nanoparticles (NPs), upon introduction to the biological systems, become wrapped by serum and cellular proteins constituting the protein corona (PC). This PC contributes largely to the NPs’ interaction with the biological systems and their subsequent functions. On the one hand, PC can decrease the efficiency of targeting by directing the NPs to the reticuloendothelial system (RES) or by masking the active targeting moieties and decreasing their ability to bind to their target receptors. On the other hand, some components of PC have offered hopes for achieving endogenous targeting.Methods: In this study, we aimed at the investigation of the role of the PC in determining the behavior of cRGDyk peptide-unconjugated and -conjugated NPs (uNPs and cNPs) exhibiting different physicochemical properties and their interaction with melanoma on in vitro and in vivo levels. Mathematical modeling has been utilized to understand the kinetics of the interaction of NPs with the tumor cells and different organs, respectively.Results: Endocytosis and exocytosis were reported to occur simultaneously for the utilized NPs. The balance was largely dependent on the NPs’ physicochemical properties and the role of the PC. In addition, distinct proteins present in the PC (illustrated in the results of the PC analysis in part I) have also determined the patterns of the NPs’ distribution in different organs and tissues of the vascular system, the RES system and the target tumot tissue. Vitronectin (VN) was found to mediate higher accumulation in integrin receptor-expressing melanoma cells, while complement 3 protein (C3) and clusterin (CLU), as an opsonin and dysopsonin, respectively, regulated the balance between the RES uptake and blood circulation.Discussion: PC, if properly modulated by tuning NPs’ physicochemical properties, can serve as a potential venue for optimum utilization of NPs in cancer therapy.Keywords: Protein corona, endogenous targeting, melanoma, endocytosis, exocytosis, biodistribution, pharmacokinetics

Aya Ahmed Sebak,1 Iman Emam Omar Gomaa,2 Aliaa Nabil ElMeshad,3 Mahmoud Hussien Farag,1 Ulrike Breitinger,4 Hans-Georg Breitinger,4 Mahmoud Hashem AbdelKader5,6 1Pharmaceutical Technology Department, Faculty of Pharmacy and Biotechnology, German University in Cairo (GUC), New Cairo City, Egypt; 2Biochemistry Department, Faculty of Pharmacy, October University for Modern Sciences and Arts (MSA), Giza, Egypt; 3Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, Cairo University, Giza, Egypt; 4Biochemistry Department, Faculty of Pharmacy and Biotechnology, German University in Cairo (GUC), New Cairo City, Egypt; 5National Institute of Laser Enhanced Sciences (NILES), Cairo University (CU), Giza, Egypt; 6European University in Egypt (EUE), New Administrative Capital, Cairo, EgyptCorrespondence: Aya Ahmed SebakFaculty of Pharmacy and Biotechnology, German University in Cairo, Cairo, EgyptTel +20 1205727787Email aya.sebak@gmail.comIman Emam Omar GomaaBiochemistry Department Faculty of Pharmacy, October University for Modern Sciences and Arts (MSA), Giza, EgyptTel +20 1275146432Email gomaa.iman@gmail.comIntroduction: Protein corona (PC) deposition on nanoparticles (NPs) in biological systems contributes to a great extent to NPs’ fates; their targeting potential, the interaction with different biological systems and the subsequent functions. PC – when properly tuned – can serve as a potential avenue for optimization of NPs’ use in cancer therapy.Methods: Poly-lactic co-glycolic acid (PLGA)-based NPs exhibiting different physicochemical properties were fabricated and characterized. The PC makeup of these NPs were qualitatively and quantitatively analyzed by Western blot and Bradford assay, respectively. The effect of PC on the release of NPs’ cargos and the intracellular uptake into B16F10 melanoma cells has been studied.Results: The composition of NPs (polymeric PLGA NPs vs lipid-polymer hybrid NPs) and the conjugation of an active targeting ligand (cRGDyk peptide) represented the major determinants of the PC makeup of NPs. The in vitro release of the loaded cargos from the NPs depended on the PC and the presence of serum proteins in the release medium. Higher cumulative release has been recorded in the presence of proteins in the case of peptide conjugated NPs, cNPs, while the unconjugated formulations, uNPs, showed an opposite pattern. NPs intracellular uptake studies revealed important roles of distinct serum and cellular proteins on the extent of NPs’ accumulation in melanoma cells. For example, the abundance of vitronectin (VN) protein from serum has been positively related to the intracellular accumulation of the NPs.Conclusion: Careful engineering of nanocarriers can modulate the recruitment of some proteins suggesting a potential use for achieving endogenous targeting to overcome the current limitations of targeted delivery of chemotherapeutic agents.Keywords: nanoparticles, active targeting, passive targeting, endogenous targeting, melanoma, protein corona, intracellular uptake

Lipid nanoparticles (LNPs) are a clinically mature technology for the delivery of genetic medicines but have limited therapeutic applications due to liver accumulation. Recently, our laboratory developed selective organ targeting (SORT) nanoparticles that expand the therapeutic applications of genetic medicines by enabling delivery of messenger RNA (mRNA) and gene editing systems to non-liver tissues. SORT nanoparticles include a supplemental SORT molecule whose chemical structure determines the LNP's tissue-specific activity. To understand how SORT nanoparticles surpass the delivery barrier of liver hepatocyte accumulation, we studied the mechanistic factors which define their organ-targeting properties. We discovered that the chemical nature of the added SORT molecule controlled biodistribution, global/apparent pK a , and serum protein interactions of SORT nanoparticles. Additionally, we provide evidence for an endogenous targeting mechanism whereby organ targeting occurs via 1) desorption of poly(ethylene glycol) lipids from the LNP surface, 2) binding of distinct proteins to the nanoparticle surface because of recognition of exposed SORT molecules, and 3) subsequent interactions between surface-bound proteins and cognate receptors highly expressed in specific tissues. These findings establish a crucial link between the molecular composition of SORT nanoparticles and their unique and precise organ-targeting properties and suggest that the recruitment of specific proteins to a nanoparticle's surface can enable drug delivery beyond the liver.