Design, synthesis and immunological evaluation of self-assembled antigenic peptides from dual-antigen targets: a broad-spectrum candidate for an effective antibreast cancer therapy

BackgroundConsidering the narrow immune response spectrum of a single epitope, and the nanoparticles (NPs) as a novel adjuvant can achieve efficient delivery of antigenic peptides safely, a nano-system (denoted as DSPE-PEG-Man@EM-NPs) based on cathepsin B-responsive antigenic peptides was designed and synthesized.MethodsHighly affinitive antigenic peptides were delivered by self-assembled NPs, and targeted erythrocyte membranes acted as a peptide carrier to improve antigenic peptides presentation and to strengthen cytotoxic T-cells reaction. Cathepsin B coupling could release antigenic peptides rapidly in dendritic cells.ResultsEvaluations showed that DSPE-PEG-Man@EM-NPs had obvious inhibitory effects towards both MCF-7 and MDA-MB-231 human breast cancer cell lines.ConclusionOverall, this strategy provides a novel strategy for boosting cytotoxic T lymphocytes response, thereby expanding the adaptation range of tumor antigenic peptides and improving the therapeutic effect of tumor immunotherapy with nanomedicine.

A Nanomule Peptide Carrier Delivers siRNA Across the Intact Blood-Brain Barrier to Attenuate Ischemic Stroke

The blood-brain barrier (BBB) hinders the distribution of therapeutics intended for treatment of neuroinflammation (NI) of the central nervous system. A twelve-amino acid peptide that transcytoses the BBB, termed MTfp, was chemically conjugated to siRNA to create a novel peptide-oligonucleotide conjugate (POC), directed to downregulate NOX4, a gene thought responsible for oxidative stress in ischemic stroke. The MTfp-NOX4 POC has the ability to cross the intact BBB and knockdown NOX4 expression in the brain. Following induction of ischemic stroke, animals pretreated with the POC exhibited significantly smaller infarcts; accompanied by increased protection against neurological deterioration and improved recovery. The data demonstrates that the MTfp can act as a nanomule to facilitate BBB transcytosis of siRNAs; where the NOX-4 specific siRNA moiety can elicit effective therapeutic knockdown of a gene responsible for oxidative stress in the central nervous system. This study is the first to conclusively demonstrate both siRNA-carrier delivery and therapeutic efficacy in any CNS disease model where the BBB remains intact and thus offers new avenues for potential treatments of oxidative stress underlying neuroinflammation in a variety of neuropathologies that are currently refractory to existing therapies.

Development of iRGD-Modified Peptide Carriers for Suicide Gene Therapy of Uterine Leiomyoma

Uterine leiomyoma (UL) is one of the most common benign tumors in women that often leads to many reproductive complications. Suicide genetherapy was suggested as a promising approach for UL treatment. In the present study, we describe iRGD ligand-conjugated cysteine-rich peptide carrier RGD1-R6 for targeted DNA delivery to αvβ3 integrin-expressing primary UL cells. The physico-chemical properties, cytotoxicity, transfection efficiency and specificity of DNA/RGD1-R6 polyplexes were investigated. TheHSV-1thymidine kinase encoding plasmid delivery to PANC-1pancreatic carcinoma cells and primary UL cells resulted in significant suicide gene therapy effects. Subsequent ganciclovir treatment decreased cells proliferative activity, induced of apoptosis and promoted cells death.The obtained results allow us to concludethatthe developed RGD1-R6 carrier can be considered a promising candidate for suicide gene therapy of uterine leiomyoma.

The Use of Ferritin as a Carrier of Peptides and Its Application for Hepcidin

Hepcidin a 25-amino-acid and highly disulfide bonded hormone, is the central regulator of iron homeostasis. In this chapter we propose ferritin as a peptide carrier to promote the association of the hybrid hepcidin/ferritin nanoparticle with a particular cell or tissue for therapeutic or diagnostic use. Indeed, human ferritin H-chain fused directly (on its 5’end) with camel mature hepcidin was cloned into the pASK-43 plus vector and expressed using BL21 (DE3) pLys E. coli strain. The transformed E.coli produced efficiently hepcidin-ferritin construct (hepcH), consisting of 213 amino acids with a molecular weight of 24 KDa. The recovered product is a ferritin exposing hepcidin on outer surface. The hepcH monomer was characterized by immunoblotting using a monoclonal antibody specific for human ferritin and a polyclonal antibody specific for hepcidin-25. The results were also confirmed by MALDI-TOF mass spectrometry. The recombinant native human ferritin and the commercial human hepcidin-25 were used as controls in this experiment. The assembly of hepcH, as an heteropolymer molecule, was performed in presence of denatured human ferritin-H and -L chains. After cysteine oxidation of the recombinant nanoparticles, cellular binding assays were performed on mammalian cells such as mouse monocyte–macrophage cell line J774, HepG2 and COS7.

Characterization of iRGD-Ligand Modified Arginine-Histidine-Rich Peptides for Nucleic Acid Therapeutics Delivery to αvβ3 Integrin-Expressing Cancer Cells

Efficient and specific delivery of nucleic acid (NA) therapeutics to tumor cells is extremely important for cancer gene therapy. Various therapeutic strategies include delivery of DNA-therapeutics such as immunostimulatory or suicide genes and delivery of siRNA-therapeutics able to silence expression of cancer-related genes. Peptides are a promising class of non-viral vehicles which are biodegradable and can efficiently condense, protect and specifically deliver NA to the cells. Here we designed arginine-histidine-rich peptide carriers consisting of an iRGD ligand to target αvβ3 integrins and studied them as vehicles for DNA and siRNA delivery to cancer cells. Combination of iRGD-modified and unmodified arginine–histidine-rich peptides during NA complexation resulted in carriers with different ligand contents. The NA-binding and protecting properties in vitro transfection efficiency and cytotoxicity of the DNA- and siRNA-polyplexes were studied and the most efficient carrier RGD1 was determined. The ability of the peptides to mediate specific intracellular uptake was confirmed inhuman cervical carcinoma (HeLa), human kidney (293T) and human pancreatic (PANC-1) cell lines with different αvβ3 integrins surface expression. By means of RGD1 carrier, efficient delivery of the herpes simplex virus (HSV-1) thymidine kinase gene to PANC-1 cells was demonstrated. Subsequent ganciclovir treatment led to a reduction of PANC-1 cells’ viability by up to 54%. Efficient RNAi-mediated down-regulation of GFP and VEGFA gene expression was achieved in MDA-MB-231-GFP+ breast cancer and EA.hy926 endothelial cells, respectively, by means of RGD1/siRNA polyplexes. Here we demonstrated that the peptide carrier RGD1 can be considered as promising candidate for development of NA therapeutics delivery systems useful in cancer gene therapy.

The Use of Electrochemical Voltammetric Techniques and High-Pressure Liquid Chromatography to Evaluate Conjugation Efficiency of Multiple Sclerosis Peptide-Carrier Conjugates

Recent studies have shown the ability of electrochemical methods to sense and determine, even at very low concentrations, the presence and quantity of molecules or analytes including pharmaceutical samples. Furthermore, analytical methods, such as high-pressure liquid chromatography (HPLC), can also detect the presence and quantity of peptides at very low concentrations, in a simple, fast, and efficient way, which allows the monitoring of conjugation reactions and its completion. Graphite/SiO2 film electrodes and HPLC methods were previously shown by our group to be efficient to detect drug molecules, such as losartan. We now use these methods to detect the conjugation efficiency of a peptide from the immunogenic region of myelin oligodendrocyte to a carrier, mannan. The HPLC method furthermore confirms the stability of the peptide with time in a simple one pot procedure. Our study provides a general method to monitor, sense and detect the presence of peptides by effectively confirming the conjugation efficiency. Such methods can be used when designing conjugates as potential immunotherapeutics in the treatment of diseases, including multiple sclerosis.

Rational Design of Hypoallergenic Vaccines: Blocking IgE-binding to Polcalcin Using Allergen-specific IgG Antibodies

Chenopodium album polcalcin (Che a 3) is characterized as a major cause of cross-reactivity inallergic patients to the Chenopodiaceae family. Therefore, the present study was conducted to develop a hypoallergenic Che a 3 derivatives as the candidate vaccine for type 1 allergy. Four derivatives were generated from Che a 3. The first was a mosaic peptide derivative computationally identified in Che a 3 which was coupled to keyhole limpet hemocyanin (KLH). The second one was a mutant Che a 3, and the other two derivatives included N- and C-terminal halves of Che a 3 that both coupled to KLH. The IgE-binding capacity of Che a 3 and its derivatives and also their ability to induce there combinant Che a 3 (rChe a 3)-specific IgG antibody, were determined using the enzyme-linked immune sorbent assay (ELISA). Moreover, the lymphopro liferative capacity of rChe a 3 or its derivatives and their pro-inflammatory cytokine response interleukin (IL)-5 and IL-13 were measured in the human peripheral blood mononuclear cells (PBMCs). Among all derivatives, the N-terminal half peptide and mosaic peptide exhibited the lowest IgE-binding capacity. In addition, in comparison to other antigens, KLH-coupled mosaic peptide induced the highest level of the recombinant Che a 3 (rChe a 3)-specific IgG antibody and ther Che a 3 specific-blocking IgG antibody in mice. Moreover, the mosaic peptide lacked lymphopro liferative capacity and down-regulated expression of pro-allergic IL-5 and IL-13 cytokines. Therefore, a peptide-carrier fusion vaccine, composed of the B-cell epitope coupled to the carrier, could be considered as one of the promising hypoallergenic vaccines to treat patients with allergy to low molecular weight allergens such as Che a 3.