Multi-Layered PLGA-PEI Nanoparticles Functionalized with TKD Peptide for Targeted Delivery of Pep5 to Breast Tumor Cells and Spheroids

Akhil K Mohan,1,2 Minsa M,3 T R Santhosh Kumar,3 G S Vinod Kumar1 1Nano Drug Delivery Systems (NDDS), Cancer Biology Division, Rajiv Gandhi Centre for Biotechnology, Thiruvananthapuram, Kerala, 695014, India; 2Research Centre, Department of Biotechnology, University of Kerala, Thiruvananthapuram, Kerala, India; 3Cancer Research Programme-1, Bio-Innovation Center (BIC), Rajiv Gandhi Centre for Biotechnology, Thiruvananthapuram, Kerala, 695014, IndiaCorrespondence: G S Vinod Kumar, Tel +91 471 2781217, Fax +91 471 2348096, Email [email protected]: Peptide-based therapy is a promising strategy for cancer treatment because of its low drug resistance. However, the major challenge is their inability to target cancer cells specifically. So, a targeted nano-delivery system that could deliver therapeutic peptides selectively to cancer cells to stimulate their action is highly desirable. This study aims to deliver the antitumor peptide, Pep5, to breast tumor cells selectively using a targeting peptide functionalised multi-layered PLGA-PEI nanoparticles.Methods: In this study, Pep5 entrapped PLGA-PEI (Pep5-PPN) dual layered nanoparticles were developed. These nanoparticles were decorated with TKD (Pep5-TPPN) on their surface for site-specific delivery of Pep5 to breast tumor cells. The particles were then characterized using various instrumental analyses. In vitro cytotoxicity of the particles was evaluated in estrogen receptor positive (ER+ve) and triple negative breast cancer (TNBC) cells. An ex vivo tumor spheroid model was used to analyze the antitumor activity of the particles.Results: Uniformly round Pep5-TPPN particles were synthesized with an average diameter of 420.8 ± 14.72 nm. The conjugation of PEI over Pep5-PLGA nanoparticles shifted the zeta potential from − 11.6 ± 2.16 mV to +20.01 ± 2.97 mV. In vitro cytotoxicity analysis proved that TKD conjugation to nanoparticles enhanced the antitumor activity of Pep5 in tested breast cancer cells. Pep5-TPPN induced cytoskeletal damage and apoptosis in the tested cells, which showed that the mechanism of action of Pep5 is conserved but potentiated. Active targeting of Pep5 suppressed the tumor growth in ex vivo spheroid models.Conclusion: A multi-layered nanoparticle functionalized with dual peptide was fabricated for active tumor targeting, which stimulated Pep5 activity to reduce the tumor growth in vitro and ex vivo.Graphical Abstract: Keywords: peptide, Pep 5, tumor, cancer, breas

Pan-active imidazolopiperazine antimalarials target the Plasmodium falciparum intracellular secretory pathway

A promising new compound class for treating human malaria is the imidazolopiperazines (IZP) class. IZP compounds KAF156 (Ganaplacide) and GNF179 are effective against Plasmodium symptomatic asexual blood-stage infections, and are able to prevent transmission and block infection in animal models. But despite the identification of resistance mechanisms in P. falciparum, the mode of action of IZPs remains unknown. To investigate, we here combine in vitro evolution and genome analysis in Saccharomyces cerevisiae with molecular, metabolomic, and chemogenomic methods in P. falciparum. Our findings reveal that IZP-resistant S. cerevisiae clones carry mutations in genes involved in Endoplasmic Reticulum (ER)-based lipid homeostasis and autophagy. In Plasmodium, IZPs inhibit protein trafficking, block the establishment of new permeation pathways, and cause ER expansion. Our data highlight a mechanism for blocking parasite development that is distinct from those of standard compounds used to treat malaria, and demonstrate the potential of IZPs for studying ER-dependent protein processing

Defining the determinants of specificity of Plasmodium proteasome inhibitors

The Plasmodium proteasome is an emerging antimalarial target due to its essential role in all the major life cycle stages of the parasite and its contribution to the establishment of resistance to artemisinin (ART)-based therapies. However, because of a similarly essential role for the host proteasome, the key property of any antiproteasome therapeutic is selectivity. Several parasite-specific proteasome inhibitors have recently been reported, however, their selectivity must be improved to enable clinical development. Here we describe screening of diverse libraries of non-natural synthetic fluorogenic substrates to identify determinants at multiple positions on the substrate that produce enhanced selectivity. We find that selection of an optimal electrophilic “warhead” is essential to enable high selectivity that is driven by the peptide binding elements on the inhibitor. We also find that host cell toxicity is dictated by the extent of coinhibition of the human β2 and β5 subunits. Using this information, we identify compounds with over 3 orders of magnitude selectivity for the parasite enzyme. Optimization of the pharmacological properties resulted in molecules that retained high potency and selectivity, were soluble, sufficiently metabolically stable and orally bioavailable. These molecules are highly synergistic with ART and can clear parasites in a mouse model of infection, making them promising leads as antimalarial drugs

Hexahydroquinolines are antimalarial candidates with potent blood-stage and transmission-blocking activity

Antimalarial compounds with dual therapeutic and transmission-blocking activity are desired as high-value partners for combination therapies. Here, we report the identification and characterization of hexahydroquinolines (HHQs) that show low nanomolar potency against both pathogenic and transmissible intra-erythrocytic forms of the malaria parasite Plasmodium falciparum. This activity translates into potent transmission-blocking potential, as shown by in vitro male gamete formation assays and reduced oocyst infection and prevalence in Anopheles mosquitoes. In vivo studies illustrated the ability of lead HHQs to suppress Plasmodium berghei blood-stage parasite proliferation. Resistance selection studies, confirmed by CRISPR-Cas9-based gene editing, identified the digestive vacuole membrane-spanning transporter PfMDR1 (P. falciparum multidrug resistance gene-1) as a determinant of parasite resistance to HHQs. Haemoglobin and haem fractionation assays suggest a mode of action that results in reduced haemozoin levels and might involve inhibition of host haemoglobin uptake into intra-erythrocytic parasites. Furthermore, parasites resistant to HHQs displayed increased susceptibility to several first-line antimalarial drugs, including lumefantrine, confirming that HHQs have a different mode of action to other antimalarials drugs for which PfMDR1 is known to confer resistance. This work evokes therapeutic strategies that combine opposing selective pressures on this parasite transporter as an approach to countering the emergence and transmission of multidrug-resistant P. falciparum malaria