ABSTRACT
BACKGROUND: Despite the high prevalence of allergic asthma, currently, avoidance of the responsible allergens, which is nearly impossible for allergens such as house dust mite (HDM), remains among the most effective treatment. Consequently, determination of the immunogenic epitopes of allergens will aid in developing a better understanding of the condition for diagnostic and therapeutic purposes. Current methods of epitope identification, however, only evaluate immunoglobulin E-epitope binding interactions, which is not directly related to epitope immunogenicity. OBJECTIVE: To determine and rank the immunogenicity of the epitopes of major HDM allergen, Der p 2. METHODS: We performed degranulation assays with RBL-SX38 cells primed using patient plasma and challenged with nanoallergens which multivalently displayed epitopes to study the relative immunogenicity of various epitopes of Der p 2. Nanoallergens were used to evaluate epitopes individually or in combination. RESULTS: When evaluated using 3 patient samples, 3 epitopes in 2 distal regions of Der p 2 were identified as highly immunogenic when presented in combination, whereas no individual epitope triggered relevant degranulation. One of the epitopes (69-DPNACHYMKCPLVKGQQY-86) was identified to be cooperatively immunogenic when combined with other epitopes. CONCLUSION: Our study highlights the importance of conformational epitopes in HDM-related allergies. This study also provides further evidence of the versatility of nanoallergens and their value for functional characterization of allergy epitopes, by ranking the Der p 2 epitopes according to immunogenicity. We believe that nanoallergens, by aiding in identification and understanding of immunogenic epitopes, will provide a better understanding of the manifestation of the allergic condition and potentially aid in developing new treatments.
Subject(s)
Antigens, Dermatophagoides , Pyroglyphidae , Allergens , Animals , Arthropod Proteins , Dust , Epitopes/chemistry , HumansABSTRACT
Here, rational engineering of doxorubicin prodrug loaded peptide-targeted liposomal nanoparticles to selectively target metastatic breast cancer cells in vivo is described. Glucose-regulated protein 78 (GRP78), a heat shock protein typically localized in the endoplasmic reticulum in healthy cells, has been identified to home to the cell surface in certain cancers, and thus has emerged as a promising therapeutic target. Recent reports indicated GRP78 to be expressed on the cell surface of an aggressive subpopulation of stem-like breast cancer cells that exhibit metastatic potential. In this study, a targeted nanoparticle formulation with a GRP78-binding peptide (Kd of 7.4 ± 1.0 µM) was optimized to selectively target this subpopulation. In vitro studies with breast cancer cell lines showed the targeted nanoparticle formulation (TNPGRP78pep) achieved enhanced cellular uptake, while maintaining selectivity over the control groups. In vivo, TNPGRP78pep loaded with doxorubicin prodrug was evaluated using a lung metastatic mouse model and demonstrated inhibition of breast cancer cell seeding to lungs down at the level of negative control groups. Combined, this study established that specific-targeting of surface GRP78 expressing a subpopulation of aggressive breast cancer cells was able to inhibit breast cancer metastasis to lungs, and underpinned the significance of GRP78 in breast cancer metastasis.
Subject(s)
Neoplasms , Prodrugs , Animals , Mice , Endoplasmic Reticulum Chaperone BiP , Membrane Proteins , Cell Line, Tumor , Glucose , Peptides , Doxorubicin/pharmacologyABSTRACT
Here, we report a CD138 receptor targeting liposomal formulation (TNP[Prodrug-4]) that achieved efficacious tumor growth inhibition in treating multiple myeloma by overcoming the dose limiting severe toxicity issues of a highly potent drug, Mertansine (DM1). Despite the promising potential to treat various cancers, due to poor solubility and pharmacokinetic profile, DM1's translation to the clinic has been unsatisfactory. We hypothesized that the optimal prodrug chemistry would promote efficient loading of the prodrug into targeted nanoparticles and achieve controlled release following endocytosis by the cancer cells, consequently, accomplish the most potent tumor growth inhibition. We evaluated four functional linker chemistries for synthesizing DM1-Prodrug molecules and evaluated their stability and cancer cell toxicity in vitro. It was determined that the phosphodiester moiety, as part of nanoparticle formulations, demonstrated most favorable characteristics with an IC50 of â¼16 nM. Nanoparticle formulations of Prodrug-4 enabled its administration at 8-fold higher dosage of equivalent free drug while remaining below maximum tolerated dose. Importantly, TNP[Prodrug-4] achieved near complete inhibition of tumor growth (â¼99% by day 10) compared to control, without displaying noticeable systemic toxicity. TNP[Prodrug-4] promises a formulation that could potentially make DM1 treatment available for wider clinical applications with a long-term goal for better patient outcomes.
Subject(s)
Maytansine , Multiple Myeloma , Nanoparticles , Prodrugs , Humans , Prodrugs/chemistry , Multiple Myeloma/drug therapy , Maytansine/therapeutic use , Maytansine/pharmacology , Nanoparticles/chemistry , Liposomes , Peptides , Cell Line, TumorABSTRACT
Development of effective targeted nanoparticle (TNP) therapeutics requires rational design of targeted and endosomolytic moieties. Nevertheless, endosomal escape of TNPs is poorly understood, relying on extrapolation of knowledge from nontargeted (NP) systems. Here, we describe how incorporation of targeting elements on endosomolytic nanoparticles alters the endosomal escape mechanism. We demonstrated that NP and TNP systems react differently to addition of precise length oligohistidines and showcase the effects of alternating spatial arrangements of targeting and endosomolytic elements. The results established that these elements act cooperatively and must be incorporated as individual moieties, rather than a single multifunctional moiety, for optimal internalization by target cells.
Subject(s)
Liposomes , Nanoparticles , Endosomes/metabolism , Liposomes/metabolism , Liposomes/pharmacology , Nanoparticles/therapeutic useABSTRACT
Endosomal escape of nanoparticles (NPs) is a weighty consideration for engineering successful nanomedicines. Although it is well-established that incorporation of histidine (His) in particle design improves endosomal escape for NPs, our understanding of its effects for ligand-targeted nanoparticles (TNPs) remains incomplete. Here, we systematically evaluated the cooperativity between targeting ligands and endosomolytic elements using liposomal TNPs with precise stoichiometric control over functional moieties (>90% loading efficiency). We synthesized endosomolytic lipid conjugates consisting of 1 to 10 consecutive His residues presented at the end of linkers between 2 to 45 repeating units of ethylene glycol (Hisn-EGm). Hisn-EGm had minimal effect on NP size (â¼115 nm) and had no significant effect on the receptor specificity of TNPs (>90% inhibition by competing peptide). We evaluated various formulations with 8 different targeting ligands relevant to two disease models. Incorporation of His1-EG8 resulted in up to â¼170- and â¼12.9-fold enhancement in intracellular accumulation relative to non-endosomolytic NP and TNP, respectively. These observations were time-dependent, targeted receptor-dependent, and showed different trends for NPs and TNPs. Further evaluation demonstrated short linkers (EG2-4) significantly enhanced nanoparticle internalization compared to EG8 or longer by up to â¼2.5-fold. Finally, rationally optimized formulation, His1-EG2-TNP, improved in vitro toxicity of a DM1 prodrug to SK-BR-3 cells by â¼4.2-fold, with IC50 â¼8.5 nM compared to â¼36 nM for no-His TNP, and >100 nM for non-targeted/no-His NP. This study uncovers an intricate relationship between endosomal escape and ligand-targeted drug delivery, as well as tunable parameters. Furthermore, our findings highlight the value of rational design and systematic analysis for optimization of multifunctional NPs.
Subject(s)
Nanoparticles , Prodrugs , Drug Delivery Systems , Endosomes , Peptides , Prodrugs/pharmacologyABSTRACT
BACKGROUND: Drug-loaded nanoparticles have established their benefits in the fight against multiple myeloma; however, ligand-targeted nanomedicine has yet to successfully translate to the clinic due to insufficient efficacies reported in preclinical studies. METHODS: In this study, liposomal nanoparticles targeting multiple myeloma via CD38 or CD138 receptors are prepared from pre-synthesized, purified constituents to ensure increased consistency over standard synthetic methods. These nanoparticles are then tested both in vitro for uptake to cancer cells and in vivo for accumulation at the tumor site and uptake to tumor cells. Finally, drug-loaded nanoparticles are tested for long-term efficacy in a month-long in vivo study by tracking tumor size and mouse health. RESULTS: The targeted nanoparticles are first optimized in vitro and show increased uptake and cytotoxicity over nontargeted nanoparticles, with CD138-targeting showing superior enhancement over CD38-targeted nanoparticles. However, biodistribution and tumor suppression studies established CD38-targeted nanoparticles to have significantly increased in vivo tumor accumulation, tumor cell uptake, and tumor suppression over both nontargeted and CD138-targeted nanoparticles due to the latter's poor selectivity. CONCLUSION: These results both highlight a promising cancer treatment option in CD38-targeted nanoparticles and emphasize that targeting success in vitro does not necessarily translate to success in vivo.
Subject(s)
ADP-ribosyl Cyclase 1/metabolism , Antibiotics, Antineoplastic/administration & dosage , Doxorubicin/administration & dosage , Drug Delivery Systems , Liposomes/metabolism , Multiple Myeloma/drug therapy , Syndecan-1/metabolism , ADP-ribosyl Cyclase 1/chemistry , Animals , Antibiotics, Antineoplastic/pharmacokinetics , Cell Line, Tumor , Doxorubicin/pharmacokinetics , Humans , Liposomes/chemistry , Male , Mice , Mice, Inbred NOD , Mice, SCID , Molecular Docking Simulation , Multiple Myeloma/metabolism , Peptides/chemistry , Peptides/metabolism , Syndecan-1/chemistry , Tissue DistributionABSTRACT
Recently, immunotherapy has emerged as a potential, possibly safer, alternative to more traditional chemotherapeutic treatments. Nevertheless, combating the tumor microenvironment (TME) and reactivating the immune system is not without complications. A recent report suggests a rationally designed supramolecular assembly to offer a solution to this problem.