Intracellular Delivery of Anti-Kirsten Rat Sarcoma Antibodies Mediated by Polymeric Micelles Exerts Strong In Vitro and In Vivo Anti-Tumorigenic Activity in Kirsten Rat Sarcoma-Mutated Cancers

Abstract

The Kirsten rat sarcoma viral oncogene (KRAS) is one of the most well-known proto-oncogenes, frequently mutated in pancreatic and colorectal cancers, among others. We hypothesized that the intracellular delivery of anti-KRAS antibodies (KRAS-Ab) with biodegradable polymeric micelles (PM) would block the overactivation of the KRAS-associated cascades and revert the effect of its mutation. To this end, PM-containing KRAS-Ab (PM-KRAS) were obtained using Pluronic F127. The feasibility of using PM for antibody encapsulation as well as the conformational change of the polymer and its intermolecular interactions with the antibodies was studied, for the first time, using in silico modeling. In vitro, encapsulation of KRAS-Ab allowed their intracellular delivery in different pancreatic and colorectal cancer cell lines. Interestingly, PM-KRAS promoted a high proliferation impairment in regular cultures of KRAS-mutated HCT116 and MIA PaCa-2 cells, whereas the effect was neglectable in non-mutated or KRAS-independent HCT-8 and PANC-1 cancer cells, respectively. Additionally, PM-KRAS induced a remarkable inhibition of the colony formation ability in low-attachment conditions in KRAS-mutated cells. In vivo, when compared with the vehicle, the intravenous administration of PM-KRAS significantly reduced tumor volume growth in HCT116 subcutaneous tumor-bearing mice. Analysis of the KRAS-mediated cascade in cell cultures and tumor samples showed that the effect of PM-KRAS was mediated by a significant reduction of the ERK phosphorylation and a decrease in expression in the stemness-related genes. Altogether, these results unprecedently demonstrate that the delivery of KRAS-Ab mediated by PM can safely and effectively reduce the tumorigenicity and the stemness properties of KRAS-dependent cells, thus bringing up new possibilities to reach undruggable intracellular targets.

Delivery Systems in Ocular Retinopathies: The Promising Future of Intravitreal Hydrogels as Sustained-Release Scaffolds

“…Thanks to representative computational methods such as molecular dynamic simulation [ 177 ], docking [ 178 ], semi-empirical quantum mechanical methods [ 179 ] and free-energy perturbation [ 180 ], among others, it is possible to achieve the rational design of HGs. Basically, through these tools it is possible to better understand how the structure of HGs can interact with a specific therapeutic agent such as a drug, peptide, protein, antibody, etc., at an atomic and molecular level [ 180 , 181 ]. With these methods, it can be possible to predict the intermolecular interactions that govern the affinity of the HG toward a specific molecule.…”

“…However, as previously mentioned, this only complies when the load of the therapeutic agent into the HG is predominated by weak interactions, for example, hydrophilic and hydrophobic interactions, van der Waals and electrostatic interactions, hydrogen bonds, and salt bridges [ 176 , 182 ]. The computational methods predict the intermolecular interactions between moieties (functional groups) from HG and moieties of the studied therapeutic agent [ 181 ]. Below we outline some basic computational methods.…”

Personalized Cancer Nanomedicine: Overcoming Biological Barriers for Intracellular Delivery of Biopharmaceuticals

“…Following administration of this formulation in a subcutaneous colon G13D‐mutant KRAS‐bearing mouse model, both tumor size reduction and diminished KRAS and ERK protein expression, was observed. [ 257 ] A different approach (no mAb) was adopted by Haley et al., which consisted in the administration of a designed ankyrin repeat protein targeting RAS‐loaded LNPs. Although intracellular delivery of the protein was confirmed by luminescence signal triggered by the formation of protein–protein complexes in a hepatocellular carcinoma model after the NPs accumulation in liver, the therapeutic response was comparable to that of the free protein in terms of tumor counts.…”

The Nanotechnology-Based Approaches against Kirsten Rat Sarcoma-Mutated Cancers

“…Over the last few years, different antibodies, peptides, nanobodies, and affimers have been designed to selectively bind to KRAS and its mutated forms in order to block its biological activity in a specific manner [ 87 , 88 , 89 , 90 , 91 , 92 , 93 ]. In order to improve their internalization into the cells, different approaches have been used.…”

“…These results demonstrate not only a strong downregulation of the RAS/MAPK pathway, but also a stemness phenotype in the cell, as demonstrated by the gene expression levels. Moreover, in an in vivo colon cancer model, they demonstrated a significant reduction in tumor growth for the animals treated with the micelles encapsulating the anti-KRAS antibody in comparison with the animals receiving the empty micelles ( Figure 5 ) [ 87 ]. This work was a clear example of how the antibodies have great potential not only for extracellular targets but also for intracellular targets, especially the ones considered undruggable or untargetable using other therapeutic options.…”