ABSTRACT
Functionalized antibodies and antibody fragments have found applications in the fields of biomedical imaging, theranostics, and antibody-drug conjugates (ADC). In addition, therapeutic and theranostic approaches benefit from the possibility to deliver more than one type of cargo to target cells, further challenging stochastic labeling strategies. Thus, bioconjugation methods to reproducibly obtain defined homogeneous conjugates bearing multiple different cargo molecules, without compromising target affinity, are in demand. Here, we describe a straightforward CRISPR/Cas9-based strategy to rapidly engineer hybridoma cells to secrete Fab' fragments bearing two distinct site-specific labeling motifs, which can be separately modified by two different sortase A mutants. We show that sequential genetic editing of the heavy chain (HC) and light chain (LC) loci enables the generation of a stable cell line that secretes a dual tagged Fab' molecule (DTFab'), which can be easily isolated. To demonstrate feasibility, we functionalized the DTFab' with two distinct cargos in a site-specific manner. This technology platform will be valuable in the development of multimodal imaging agents, theranostics, and next-generation ADCs.
Subject(s)
Clustered Regularly Interspaced Short Palindromic Repeats , Hybridomas/chemistry , Immunoglobulin Fab Fragments/chemistry , Antibodies, Monoclonal/chemistry , Immunoconjugates/chemistry , Stochastic ProcessesABSTRACT
High toxicity caused by chemotherapeutic drugs and the acquisition of drug resistance by cancer cells are the major drawbacks in cancer therapy. A promising approach to overcome the posed barriers is conjugating tumor-homing peptides to drugs or nanocarriers. Such high-affinity peptides can specifically target surface markers overexpressed by cancer cells, ensuring a rapid and cancer-specific uptake of the drugs. Since prostate-specific membrane antigen (PSMA) is overexpressed by aggressive prostate cancer cells, targeting this surface protein with peptide conjugates can lead to the development of effective strategies against prostate cancer. In this study, we aimed to determine which PSMA-binding peptide among peptides 563, 562 and 9-mer, show the highest selectivity towards PSMA using 22Rv1 prostate cancer cells, a cell line with moderate PSMA levels. Tumor-homing peptides were synthesized by fluorenylmethoxycarbonyl-based solid-phase peptide synthesis (Fmoc-SPPS) strategy, and evaluated for their prostate cancer cell-specific targeting efficiencies by flow cytometry. Our results showed that the PSMA-binding capacity of peptide 563 was superior to those of 562, 9-mer, and 5-mer; therefore, can be utilized as a potent-targeting agent not only in the treatment of high PSMA positive but also moderate PSMA positive prostate cancer tumors.
Subject(s)
Peptides/administration & dosage , Prostatic Neoplasms/drug therapy , Prostatic Neoplasms/metabolism , Animals , Antigens, Surface/genetics , Antigens, Surface/metabolism , Cell Line, Tumor , Drug Evaluation, Preclinical , Glutamate Carboxypeptidase II/genetics , Glutamate Carboxypeptidase II/metabolism , Humans , Male , Mice , Peptides/chemical synthesis , Prostatic Neoplasms/geneticsABSTRACT
The second mitochondria-derived activator of caspase (Smac/DIABLO) is a pro-apoptotic protein that released from mitochondria into the cytosol when cells undergo apoptosis. Smac promotes caspase activation by binding the inhibitors of apoptosis proteins (IAP), particularly XIAP and eliminating their inhibitory activity. Although the seven N-terminal amino acids AVPIAQK (SmacN7) of Smac protein is able to elicit an anticancer response by itself, it is neither cell-permeable nor stable in the cellular environment. Thus, the use of SmacN7 derivatives and mimetics is an alluring field for cancer therapy. In this study, heptamer Smac peptide was fused to a well-known octaarginine cell-penetrating peptide for promoting its intracellular access. Both therapeutic Smac part and cell-penetrating octaarginine parts of the peptide sequence constrained in a cyclic structure so as to enhance the apoptosis-inducing potential of the SmacN7 peptide. Biological assays interestingly showed that cyclic peptides P4, P5 and P7 gave rise to a significant level of cytotoxicity and apoptosis mediated cell death in multiple myeloma tumor cells (MM) comparing to linear peptide.
Subject(s)
Antineoplastic Agents , Apoptosis/drug effects , Cell-Penetrating Peptides , Multiple Myeloma/drug therapy , Oligopeptides , Peptides , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Cell Line, Tumor , Cell-Penetrating Peptides/chemical synthesis , Cell-Penetrating Peptides/chemistry , Cell-Penetrating Peptides/pharmacology , Humans , Multiple Myeloma/metabolism , Multiple Myeloma/pathology , Oligopeptides/chemical synthesis , Oligopeptides/chemistry , Oligopeptides/pharmacology , Peptides/chemical synthesis , Peptides/chemistry , Peptides/pharmacology , Structure-Activity RelationshipABSTRACT
This study focuses on creating a specialized nanogel for targeted drug delivery in cancer treatment, specifically targeting prostate cancer. This nanogel (referred to as SGK 636/Peptide 563/PEtOx nanogel) is created using hydrophilic poly(2-ethyl-2-oxazoline) (PEtOx) through a combination of living/cationic ring-opening polymerization (CROP) and alkyne-azide cycloaddition (CuAAC) "click" chemical reactions. A fluorescent probe (BODIPY) is also conjugated with the nanogel to monitor drug delivery. The characterizations through 1 H-NMR, and FT-IR, SEM, TEM, and DLS confirm the successful production of uniform, and spherical nanogels with controllable sizes (100 to 296 nm) and stability in physiological conditions. The biocompatibility of nanogels is evaluated using MTT cytotoxicity assays, revealing dose-dependent cytotoxicity. Drug-loaded nanogels exhibited significantly higher cytotoxicity against cancer cells in vitro compared to drug-free nanogels. Targeting efficiency is examined using both peptide-conjugated and peptide-free nanogels, with the intracellular uptake of peptide 563-conjugated nanogels by tumor cells being 60-fold higher than that of nanogels without the peptide. The findings suggest that the prepared nanogel holds great potential for various drug delivery applications due to its ease of synthesis, tunable functionality, non-toxicity, and enhanced intracellular uptake in the tumor region.
Subject(s)
Drug Delivery Systems , Polyethyleneimine , Prostatic Neoplasms , Humans , Male , Nanogels , Spectroscopy, Fourier Transform Infrared , Polyethylene Glycols/chemistry , Prostatic Neoplasms/drug therapy , Peptides/pharmacology , Drug Carriers/chemistryABSTRACT
The equipping of nanoparticles with the peptide moiety recognizing a particular receptor, enables cell or tissue-specific targeting, therefore the optimization of the targeted nanoparticles is a key factor in the formulation design process. In this paper, we report the optimization concept of Doxorubicin encapsulating PEtOx-b-PLA polymersome formulation equipped with Peptide18, which is a breast cancer recognizing tumor homing peptide, and the unveiling of the cell-specific delivery potential. The most dominant formulation parameters, which are the polymer to Doxorubicin mass ratio (w/w) and the aqueous to organic phase ratio (v/v), were optimized using Central Composite Design (CCD) based Response Surface Methodology. The characteristics of optimum polymersome formulation were determined as the hydrodynamic diameter of 146.35 nm, the PDI value of 0.136, and the encapsulation efficiency of 57.11% and TEM imaging, which are in agreement with the DLS data, showed the spherical morphology of the polymersomes. In order to demonstrate the breast cancer-specific delivery of targeted polymersomes, the flow cytometry and confocal microscopy analyses were carried out. The targeted polymersomes were accumulated 8 times higher in AU565 cells compared to MCF10A cells and the intracellular Doxorubicin was almost 10 times higher in AU565 cells. The CCD-mediated optimized targeted polymersomes proposed in this report holds the promise of targeted therapy for breast cancer and can be potentially used for the development of novel treatments.