Ultrasound-Triggered Liposomes Encapsulating Quantum Dots as Safe Fluorescent Markers for Colorectal Cancer

Quantum dots (QDs) are a promising tool to detect and monitor tumors. However, their small size allows them to accumulate in large quantities inside the healthy cells (in addition to the tumor cells), which increases their toxicity. In this study, we synthesized stealth liposomes encapsulating hydrophilic graphene quantum dots and triggered their release with ultrasound with the goal of developing a safer and well-controlled modality to deliver fluorescent markers to tumors. Our results confirmed the successful encapsulation of the QDs inside the core of the liposomes and showed no effect on the size or stability of the prepared liposomes. Our results also showed that low-frequency ultrasound is an effective method to release QDs encapsulated inside the liposomes in a spatially and temporally controlled manner to ensure the effective delivery of QDs to tumors while reducing their systemic toxicity.

Application of Box–Behnken Design and Desirability Function in the Development and Optimization of Stealth Liposomes of Microtubule Inhibitor

Aims: The aim of the present study was to develop and optimize a Stealth Liposomal Drug Delivery System of microtubule inhibitor using Box–Behnken Design and Desirability function. Study Design: Development and Optimization of Stealth Liposomes. Place and Duration of Study: The study was carried out in the Department of Pharmacy, Annamalai University, between September 2020 and May 2021. Methodology: Stealth Liposomes were prepared by the thin-film hydration method (TFH). The formulation was optimized using Box – Behnken design to study the effect of independent variables, Amount of Egg Phosphatidylcholine (X1), Amount of Cholesterol (X2), and Amount of DSPE-PEG 2000(X3) on dependent variables Entrapment Efficiency (Y1) and In-vitro drug release (Y2). Results: Entrapment efficiency of the Stealth Liposomes ranges from 56.35 to 84.25%and in-vitro release ranges from 62.38 to 94.26%. The optimized formulation was found using the desirability function to get maximum entrapment with maximum drug release. The optimized formulation showed entrapment efficiency of 80.46% and in-vitro release of 90.11%. Conclusion: Stealth Liposomal Drug Delivery System for microtubule inhibitor was successfully developed and optimized using desirability function in Design Expert software by a three-factor, three level Box – Behnken design.

PEGylated DPPC/Anti-SNAP25 Antibody Targeted Liposomes: From Langmuir Monolayer Study to Formulations

Background: Molecule compatibility is an important factor to be considered before preparing antibody-targeted liposomes, stealth-liposomes, and stealth antibody-targeted liposomes. Objective: To determine the intermolecular interaction of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamide-N-[methoxy(polyethyleneglycol)-2000] (ammonium salt), DOPE PEG2000 and Anti-SNAP25 (AS25) in 1,2-dipalmitoyl-sn-glycero-3-phosphorylcholine (DPPC) monolayer, and their liposomes. Methods: In this study, DPPC was used to create a monolayer mimicking the half membrane of liposomes to investigate its interactions with a polyclonal antibody, AS25, and DOPE PEG2000, respectively based on Langmuir-Blodgett (LB) techniques. The surface morphology of DPPC— AS25 and DPPC— DOPE PEG2000— AS25 bilayers were also imaged and analyzed by using atomic force microscopy (AFM) to support the LB findings. The LB findings were then utilized as a reference to prepare DPPC liposomes in this work. Results: The best mole ratio of DPPCDOPE PEG2000, determined to be 50 to 1, was used to study the interaction with the polyclonal antibody AS25. The free energy of mixing (〖Δ G〗_mix) of DPPC— DOPE PEG2000—AS25 was more negative than DPPC— AS25 in the entire investigated ranges, indicating that the ternary mixture of DPPC— DOPE PEG2000— AS25 was more compatible than the binary mixture of DPPC— AS25. The presence of DOPE PEG2000 in DPPC— AS25 increased the fluidity of the membrane, which resulted in a greater interaction of AS25 with DPPC. Conclusion: The constant values of particle size and zeta potential measurements of DPPC— DOPE PEG2000— AS25 liposomes showed agreement with the LB findings, indicating that LB is a good technique to predict precise liposomal formulations.

Ferri–Liposomes: Preformulation and Selective Cytotoxicity against A549 Lung Cancer Cells

Liposomes have become successful nanostructured systems used in clinical practices. These vesicles are able to carry important drug loadings with noteworthy stability. The aim of this work was to develop iron oxide-loaded stealth liposomes as a prospective alternative for the treatment of lung cancer. In this study, citric acid iron oxide nanoparticles (IONPs-Ac) were synthesized and encapsulated in stealth liposomes. Their cytotoxicity and selectivity against lung tumor cells were assessed. Stealth liposomal vesicles, with relevant content of IONPs-Ac, named ferri–liposomes (SL-IONPs-Ac), were produced with an average size of 200 nm. They displayed important cytotoxicity in a human lung cancer cells model (A549 cells), even at low concentrations, whereas free IONPs-Ac displayed adequate biocompatibility. Nevertheless, the treatment at the same concentration of ferri–liposomes against HEK-293 cells, a normal human cell lineage, was not significantly cytotoxic, revealing a probable lung tumor selectiveness of the fabricated formulation. Furthermore, from the flow cytometry studies, it was possible to infer that ferri–liposomes were able to induce A549 tumor cells death through apoptosis/ferroptosis processes, evidenced by a significant reduction of the mitochondrial membrane potential.

CRISPR/Cas9-loaded stealth liposomes effectively cleared established HPV16-driven tumours in syngeneic mice

Gene-editing has raised the possibility of being able to treat or cure cancers, but key challenges remain, including efficient delivery, in vivo efficacy, and its safety profile. Ideal targets for cancer therapy are oncogenes, that when edited, cause cell death. Here, we show, using the human papillomavirus (HPV) type 16 cancer cell line TC1, that CRISPR/Cas9 targeting the E7 oncogene and packaged in PEGylated liposomes cleared established tumours in immunocompetent mice. Treatment caused no significant toxicity in the spleen or liver. An ideal therapeutic outcome would be the induction of an immunogenic cell death (ICD), such that recurrent tumours would be eliminated by the host immune system. We show here for the first time that CRISPR/Cas9-mediated cell death via targeting E7 did not result in ICD. Overall, our data show that in vivo CRISPR/Cas targeting of oncogenes is an effective treatment approach for cancer.