RESUMO
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.
Assuntos
Neoplasias , Pró-Fármacos , Animais , Camundongos , Chaperona BiP do Retículo Endoplasmático , Proteínas de Membrana , Linhagem Celular Tumoral , Glucose , Peptídeos , Doxorrubicina/farmacologiaRESUMO
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.
Assuntos
Maitansina , Mieloma Múltiplo , Nanopartículas , Pró-Fármacos , Humanos , Pró-Fármacos/química , Mieloma Múltiplo/tratamento farmacológico , Maitansina/uso terapêutico , Maitansina/farmacologia , Nanopartículas/química , Lipossomos , Peptídeos , Linhagem Celular TumoralRESUMO
Although IL-9 has potent anti-tumor activity in adoptive cell transfer therapy, some models suggest that it can promote tumor growth. Here, we show that IL-9 signaling is associated with poor outcomes in patients with various forms of lung cancer, and is required for lung tumor growth in multiple mouse models. CD4+ T cell-derived IL-9 promotes the expansion of both CD11c+ and CD11c- interstitial macrophage populations in lung tumor models. Mechanistically, the IL-9/macrophage axis requires arginase 1 (Arg1) to mediate tumor growth. Indeed, adoptive transfer of Arg1+ but not Arg1- lung macrophages to Il9r-/- mice promotes tumor growth. Moreover, targeting IL-9 signaling using macrophage-specific nanoparticles restricts lung tumor growth in mice. Lastly, elevated expression of IL-9R and Arg1 in tumor lesions is associated with poor prognosis in lung cancer patients. Thus, our study suggests the IL-9/macrophage/Arg1 axis is a potential therapeutic target for lung cancer therapy.
Assuntos
Interleucina-9 , Neoplasias Pulmonares , Macrófagos , Animais , Carcinogênese/metabolismo , Interleucina-9/genética , Interleucina-9/metabolismo , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Macrófagos/metabolismo , Macrófagos/patologia , Macrófagos Alveolares/metabolismo , CamundongosRESUMO
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.
Assuntos
Nanopartículas , Pró-Fármacos , Sistemas de Liberação de Medicamentos , Endossomos , Peptídeos , Pró-Fármacos/farmacologiaRESUMO
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.
Assuntos
ADP-Ribosil Ciclase 1/metabolismo , Antibióticos Antineoplásicos/administração & dosagem , Doxorrubicina/administração & dosagem , Sistemas de Liberação de Medicamentos , Lipossomos/metabolismo , Mieloma Múltiplo/tratamento farmacológico , Sindecana-1/metabolismo , ADP-Ribosil Ciclase 1/química , Animais , Antibióticos Antineoplásicos/farmacocinética , Linhagem Celular Tumoral , Doxorrubicina/farmacocinética , Humanos , Lipossomos/química , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Simulação de Acoplamento Molecular , Mieloma Múltiplo/metabolismo , Peptídeos/química , Peptídeos/metabolismo , Sindecana-1/química , Distribuição TecidualRESUMO
B cell malignancies, such as B cell leukemia and lymphoma, have CD22 overexpression with â¼7% of patients. A short CD22 binding peptide (PV3) with a moderate affinity (Kd â¼ 9 µM) was identified by screening multiple peptide candidates determined through analysis of CD22-epratuzumab complex crystal structure. PV3 binding specificity was confirmed via competitive binding inhibition, then was used as the targeting moiety on CD22-targeted liposomal nanoparticle (TNPPV3) formulations. To maximize the potential therapeutic outcome of TNPPV3 formulation, nanoparticle design parameters, such as peptide hydrophilicity, ethylene glycol linker length, valency, and particle size were optimized for maximum selective cellular uptake by CD22+ malignant cancer cells. The effects of altering design parameters one at a time on TNP uptake were evaluated using flow cytometry, and the optimal parameters for TNPPV3 were determined to be 8% peptide density, EG18 linker, and 3 lysines of 100 nm nanoparticles. This optimally designed TNPPV3 achieved â¼4 and 40-fold enhancement of cellular uptake by CD22+ Raji cells over CD22- Jurkat and MOLT-4 cells, respectively, demonstrating selectivity for malignant cells with CD22 overexpression. Overall, this study establishes PV3 to be CD22 binding peptide with proven effectiveness as a targeting element. In future, the optimal TNPPV3 formulation will potentially achieve maximal in vivo therapeutic outcomes by efficiently targeting CD22+ blood cancer cells in vivo.
Assuntos
Linfoma de Células B/metabolismo , Nanopartículas/metabolismo , Peptídeos/metabolismo , Lectina 2 Semelhante a Ig de Ligação ao Ácido Siálico/metabolismo , Anticorpos Monoclonais Humanizados/química , Anticorpos Monoclonais Humanizados/metabolismo , Antineoplásicos/química , Antineoplásicos/metabolismo , Linhagem Celular Tumoral , Desenho de Fármacos , Endocitose , Humanos , Lipossomos/química , Lipossomos/metabolismo , Linfoma de Células B/patologia , Nanopartículas/química , Peptídeos/químicaRESUMO
Here, we report rationally engineered peptide-targeted liposomal doxorubicin nanoparticles that have an enhanced selectivity for HER2-positive breast tumor cells with high purity, reproducibility, and precision in controlling stoichiometry of targeting peptides. To increase HER2-positive tumor cell selective drug delivery, we optimized the two most important design parameters, peptide density and linker length, via systematic evaluations of their effects on both in vitro cellular uptake and in vivo tumor accumulation and cellular uptake. The optimally designed nanoparticles were finally evaluated for their tumor inhibition efficacy using in vivo MMTV-neu transplantation mouse model. In vitro, we demonstrated that ~1% peptide density and EG8 linker were optimal parameters for targeted nanoparticle formulations to enhance HER2-positive cancer cellular uptake while preventing non-selectivity. In vivo results demonstrated that at 0.5% peptide density, enhancement of tumor cell uptake over non-targeted nanoparticles was ~2.7 fold and ~3.4 fold higher for targeted nanoparticles with EG8 and EG18 linker, respectively, while their accumulation levels at tumor tissue were similar to the non-targeted nanoparticles. These results were consistent with in vivo efficacy outcomes that ~90% tumor growth inhibition was achieved by Dox-loaded HER2 receptor targeted nanoparticles, TNPHER2pep, over control while all nanoparticle formulations minimized overall systemic toxicity relative to free Dox. This study highlights the significance of understanding and optimizing the effects of liposomal nanoparticle design parameters for enhancement of tumor selectivity to achieve improved in vivo therapeutic outcomes.
Assuntos
Neoplasias da Mama , Nanopartículas , Animais , Neoplasias da Mama/tratamento farmacológico , Linhagem Celular Tumoral , Doxorrubicina/uso terapêutico , Sistemas de Liberação de Medicamentos , Feminino , Humanos , Camundongos , Peptídeos/uso terapêutico , Reprodutibilidade dos TestesRESUMO
Despite ligand-targeted liposomes long garnering interest as drug delivery vehicles for cancer therapeutics, inconsistency in successful outcomes have hindered their translation into the clinic. This is in part due to discrepancies between in vitro design evaluations and final in vivo outcomes. By employing a multifaceted synthetic strategy to prepare peptide-targeted nanoparticles of high purity, reproducibility, and with precisely controlled quantity of functionalities, we systematically evaluated the individual roles that peptide-linker length, peptide hydrophilicity, peptide density, and nanoparticle size play on cancer cell uptake and tumor targeting both in vitro and in vivo, and how the results correlated and contrasted. These parameters were analyzed using a VLA-4-targeted liposome system in a multiple myeloma mouse xenograft model to evaluate in vivo biodistribution and tumor cell uptake. The results showed that using in vitro models to optimize targeted-nanoparticles for maximum cellular uptake was helpful in narrowing down the particle characteristics. However, in vitro optimization fell short of achieving enhanced results in animal models, rather had negative consequences for in vivo targeting. This outcome is not surprising considering that the receptor being targeted is also present on healthy lymphocytes and increasing targeting peptide valency on particle surfaces results in an increase in non-selective, off-target binding to healthy cells. Hence, further optimization using in vivo models was absolutely necessary, through which we were able to increase the uptake of peptide-targeted liposomes by cancerous cells overexpressing VLA-4 to 15-fold over that of non-targeted liposomes in vivo. The results highlighted the importance of creating a comprehensive understanding of the effect of each liposome design parameter on multifactorial biological endpoints including both in vitro and in vivo in determining the therapeutic potential of peptide-targeted liposomes.
Assuntos
Integrina alfa4beta1/antagonistas & inibidores , Mieloma Múltiplo/tratamento farmacológico , Nanopartículas/administração & dosagem , Peptídeos/administração & dosagem , Animais , Linhagem Celular Tumoral , Modelos Animais de Doenças , Lipossomos , Camundongos SCID , Mieloma Múltiplo/metabolismo , Nanopartículas/química , Peptídeos/químicaRESUMO
Drug allergies occur when hapten-like drug metabolites conjugated to serum proteins, through their interactions with specific IgE, trigger allergic reactions that can be life threatening. A molecule termed covalent heterobivalent inhibitor (cHBI) was designed to specifically target drug hapten-specific IgE to prevent it from binding drug-haptenated serum proteins. cHBI binds the two independent sites on a drug hapten-specific Ab and covalently conjugates only to the specific IgE, permanently inhibiting it. The cHBI design was evaluated via ELISA to measure cHBI-IgE binding, degranulation assays of rat basophil leukemia cells for in vitro efficacy, and mouse models of ear swelling and systemic anaphylaxis responses for in vivo efficacy. The cHBI design was evaluated using two separate models: one specific to inhibit penicillin G-reactive IgE and another to inhibit IgE specific to a model compound, dansyl. We show that cHBI conjugated specifically to its target Ab and inhibited degranulation in cellular degranulation assays using rat basophil leukemia cells. Furthermore, cHBIs demonstrated in vivo inhibition of allergic responses in both murine models. We establish the cHBI design to be a versatile platform for inhibiting hapten/IgE interactions, which can potentially be applied to inhibit IgE-mediated allergic reactions to any drug/small-molecule allergy.
Assuntos
Anafilaxia/prevenção & controle , Basófilos/imunologia , Hipersensibilidade a Drogas/tratamento farmacológico , Naftalenos/metabolismo , Alérgenos/imunologia , Anafilaxia/etiologia , Animais , Complexo Antígeno-Anticorpo/imunologia , Degranulação Celular , Linhagem Celular , Modelos Animais de Doenças , Hipersensibilidade a Drogas/complicações , Ensaio de Imunoadsorção Enzimática , Epitopos/metabolismo , Feminino , Haptenos/imunologia , Humanos , Imunoglobulina E/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Naftalenos/síntese química , Penicilinas/imunologia , Ligação Proteica , RatosRESUMO
Targeted liposomal nanoparticles are commonly used drug delivery vehicles for targeting cancer cells that overexpress a particular cell surface receptor. However, typical target receptors are also expressed at variable levels in healthy tissue, leading to non-selective targeting and systemic toxicity. Here, we demonstrated that the selectivity of peptide-targeted liposomes for their target cells can be significantly enhanced by employing a dual-receptor targeted approach to simultaneously target multiple tumor cell surface receptors. The dual-receptor targeted approach can be tuned to create cooperativity in binding only for the cancer cells, therefore leaving the healthy cells and tissue unharmed. We evaluated this strategy in a multiple myeloma disease model where the liposomes were functionalized with two distinct peptide antagonists to target VLA-4 and LPAM-1, two receptors with increasing relevance in multiple myeloma. By employing a multifaceted strategy to synthesize dual-receptor targeted liposomes with high purity, reproducibility, and precisely controlled stoichiometry of functionalities, we identified optimal design parameters for enhanced selectivity via systematic analysis. Through control of the liposomal formulation and valency of each targeting peptide, we identified that the optimal dual-receptor targeted liposome consisted of a peptide density of 0.75% VLA4pep and 1% LPAM1pep, resulting in an 8-fold and 12-fold increased cellular uptake over VLA-4 and LPAM-1 single targeted liposomes respectively. This formulation resulted in a cooperative ratio of 4.3 and enhanced uptake for myeloma cells that simultaneously express both VLA-4 and LPAM-1 receptors, but displayed no increase in uptake for cells that express only one or neither of the receptors, resulting in a 28-fold selectivity of the dual-targeted liposomes for cells displaying both targeted receptors over cells displaying neither receptor. These results demonstrated that through refined design and well-characterized nanoparticle formulations, dual-receptor targeted liposomes have the potential to improve cancer therapy by providing enhanced selectivity over conventional single-receptor targeted approaches.
Assuntos
Integrina alfa4beta1 , Integrinas , Nanopartículas , Proteínas de Neoplasias , Neoplasias , Peptídeos , Humanos , Integrina alfa4beta1/antagonistas & inibidores , Integrina alfa4beta1/metabolismo , Integrinas/antagonistas & inibidores , Integrinas/metabolismo , Células Jurkat , Lipossomos , Nanopartículas/química , Nanopartículas/uso terapêutico , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Neoplasias/patologia , Peptídeos/química , Peptídeos/farmacologiaAssuntos
Alérgenos/imunologia , Antineoplásicos/efeitos adversos , Carboplatina/efeitos adversos , Hipersensibilidade a Drogas/diagnóstico , Nanopartículas , Oxaliplatina/efeitos adversos , Idoso , Linhagem Celular , Dessensibilização Imunológica , Hipersensibilidade a Drogas/etiologia , Hipersensibilidade a Drogas/imunologia , Hipersensibilidade a Drogas/terapia , Feminino , Humanos , Imunoglobulina E/imunologia , Lipossomos , Masculino , Pessoa de Meia-Idade , Testes CutâneosRESUMO
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.
Assuntos
Imunoterapia , Ativação de Macrófagos , Neoplasias/terapia , Animais , Neoplasias/imunologiaRESUMO
Small dimensions of gold nanoparticles (AuNPs) necessitate antibodies to be immobilized in an oriented fashion in order to conserve their antigen binding activity for proper function. In this study, we used the previously described UV-NBS method to site-specifically incorporate a thioctic acid (TA) functionality into antibodies at the conserved nucleotide-binding site (NBS). Modified antibodies were immobilized on the AuNP surface in an oriented manner utilizing the newly incorporated TA functionality while maintaining the antibody structure and activity. The resulting antibody functionalized AuNPs via the UV-NBS method demonstrated significantly enhanced antigen detection capabilities and improved antigen detection sensitivity with a high level of selectivity when compared to other commonly used AuNP functionalization methods. Our results demonstrate that the limit of detection (LOD) for AuNPs functionalized via the UV-NBS method was 55 pM PSA, which is 40, 851, and 5873-fold improved over the other immobilization methods: EDC-NHS, thiol reduction, and ionic interaction, respectively. Consequently, the UV-NBS method provides a universal, site-specific functionalization method that generates highly sensitive and more stable antibody functionalized AuNPs which are amenable to any available detection and treatment assay with potential significant implications.
Assuntos
Anticorpos/química , Difusão Dinâmica da Luz , Ouro , Nanopartículas Metálicas/química , Antígeno Prostático Específico/análise , Humanos , MasculinoRESUMO
Degranulation caused by type I hypersensitivity (allergies) is a complex biophysical process, and available experimental models for studying relevant immunoglobulin E binding epitopes on allergen proteins lack the ability to adequately evaluate, rank, and associate these epitopes individually and with each other. In this study, we propose a new allergy model system for studying potential allergen epitopes using nanoallergens, liposomes modified to effectively display IgE binding epitopes/haptens. By utilizing the covalently conjugated lipid tails on two hapten molecules (dinitrophenol and dansyl), hapten molecules were successfully incorporated into liposomes with high precision to form nanoallergens. Nanoallergens, with precisely controlled high-particle valency, can trigger degranulation with much greater sensitivity than commonly used bovine serum albumin conjugates. In rat basophil leukemia cell experiments, nanoallergens with only 2% hapten loading were able to trigger degranulation in vitro at concentrations as low as 10 pM. Additionally, unlike bovine serum albumin-hapten conjugates, nanoallergens allow exact control over particle size and valency. By varying the nanoallergen parameters such as size, valency, monovalent affinity of hapten, and specific IgE ratios, we exposed the importance of these variables on degranulation intensity while demonstrating nanoallergens' potential for evaluating both high- and low-affinity epitopes. The data presented in this article establish nanoallergen platform as a reliable and versatile allergy model to study and evaluate allergen epitopes in mast cell degranulation.
Assuntos
Alérgenos/química , Alérgenos/imunologia , Degranulação Celular/imunologia , Epitopos , 2,4-Dinitrofenol/imunologia , Animais , Linhagem Celular , Haptenos/imunologia , Imunoconjugados/química , Imunoglobulina E/metabolismo , Lipídeos/química , Mastócitos/imunologia , Nanoestruturas/química , Tamanho da Partícula , Fosfatidilcolinas/imunologia , RatosRESUMO
Here, we report the synthesis and evaluation of dual drug-loaded nanoparticles as an effective means to deliver carfilzomib and doxorubicin to multiple myeloma tumor cells at their optimal synergistic ratio. First, various molar ratios of carfilzomib to doxorubicin were screened against multiple myeloma cell lines to determine the molar ratio that elicited the greatest synergy using the Chou-Talalay method. The therapeutic agents were then incorporated into liposomes at the optimal synergistic ratio of 1:1 to yield dual drug-loaded nanoparticles with a narrow size range of 115 nm and high reproducibility. Our results demonstrated that the dual drug-loaded liposomes exhibited synergy in vitro and were more efficacious in inhibiting tumor growth in vivo than a combination of free drugs, while at the same time reducing systemic toxicity. Taken together, this study presents the synthesis and preclinical evaluation of dual drug-loaded liposomes containing carfilzomib and doxorubicin for enhanced therapeutic efficacy to improve patient outcome in multiple myeloma. Mol Cancer Ther; 15(7); 1452-9. ©2016 AACR.
Assuntos
Doxorrubicina/administração & dosagem , Lipossomos , Nanopartículas , Oligopeptídeos/administração & dosagem , Animais , Linhagem Celular Tumoral , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Doxorrubicina/química , Combinação de Medicamentos , Composição de Medicamentos , Avaliação Pré-Clínica de Medicamentos , Sinergismo Farmacológico , Humanos , Lipossomos/química , Camundongos , Estrutura Molecular , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/patologia , Nanopartículas/química , Oligopeptídeos/química , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Oriented immobilization of antibodies and antibody fragments has become increasingly important as a result of the efforts to reduce the size of diagnostic and sensor devices to miniaturized dimensions for improved accessibility to the end-user. Reduced dimensions of sensor devices necessitate the immobilized antibodies to conserve their antigen binding activity for proper operation. Fab fragments are becoming more commonly used in small-scaled diagnostic devices due to their small size and ease of manufacture. In this study, we used the previously described UV-NBS(Biotin) method to functionalize Fab fragments with IBA-EG11-Biotin linker utilizing UV energy to initiate a photo-cross-linking reaction between the nucleotide binding site (NBS) on the Fab fragment and IBA-Biotin molecule. Our results demonstrate that immobilization of biotinylated Fab fragments via UV-NBS(Biotin) method generated the highest level of immobilized Fab on surfaces when compared to other typical immobilization methods while preserving antigen binding activity. UV-NBS(Biotin) method provided 432-fold, 114-fold, and 29-fold improved antigen detection sensitivity than physical adsorption, NHS-Biotin, and ε-NH3(+), methods, respectively. Additionally, the limit of detection (LOD) for PSA utilizing Fab fragments immobilized via UV-NBS(Biotin) method was significantly lower than that of the other immobilization methods, with an LOD of 0.4 pM PSA. In summary, site-specific biotinylation of Fab fragments without structural damage or loss in antigen binding activity provides a wide range of application potential for UV-NBS immobilization technique across numerous diagnostic devices and nanotechnologies.
Assuntos
Anticorpos Imobilizados/química , Anticorpos Imobilizados/imunologia , Antígenos/análise , Antígenos/imunologia , Biotinilação , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/imunologia , Nucleotídeos/imunologia , Sítios de Ligação , Estrutura Molecular , Nucleotídeos/químicaRESUMO
Ligand-targeted liposomes are increasingly used as drug delivery carriers for cancer therapy, yet have not consistently produced successful outcomes. Here, we demonstrated the significant enhancement in cellular uptake of peptide-targeted liposomes by simultaneously increasing the hydrophilicity of the targeting peptide, optimizing the EG peptide-linker length, and using appropriate peptide surface density. We analyzed these parameters in a HER2-overexpressing breast cancer model system where the liposomes were functionalized with one of four distinct HER2-antagonist peptides to evaluate cellular uptake. Our results demonstrated that including a short oligolysine chain adjacent to the targeting peptide sequence effectively improved cellular uptake -6-10 fold when using an EG6-EG18 linker depending on the selected antagonist peptide. Uptake efficiency reached a maximum and a plateau with -2% peptide density with higher observed sensitivity at lower peptide densities for the more hydrophilic peptides. Taken together, these findings demonstrated the importance of optimizing liposome design for improved cellular uptake.
Assuntos
Neoplasias da Mama/metabolismo , Lipossomos/química , Nanocápsulas/química , Peptídeos/farmacocinética , Fosfatidiletanolaminas/química , Polietilenoglicóis/química , Receptor ErbB-2/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Reagentes de Ligações Cruzadas/química , Etilenoglicol/química , Feminino , Humanos , Interações Hidrofóbicas e Hidrofílicas , Teste de Materiais , Nanocápsulas/ultraestrutura , Tamanho da Partícula , Peptídeos/químicaRESUMO
Carfilzomib, a recently FDA-approved proteasome inhibitor, has remarkable anti-myeloma (MM) activity. However, its effectiveness is limited by associated severe side-effects, short circulation half-life, and limited solubility. Here, we report the engineering of liposomal carfilzomib nanoparticles to overcome these problems and enhance the therapeutic efficacy of carfilzomib by increasing tumoral drug accumulation while decreasing systemic toxicity. In our design, carfilzomib was loaded into the bilayer of liposomes to yield stable and reproducible liposomal nanoparticles. Liposomal carfilzomib nanoparticles were efficiently taken up by MM cells, demonstrated proteasome inhibition, induced apoptosis, and exhibited enhanced cytotoxicity against MM cells. In vivo, liposomal carfilzomib demonstrated significant tumor growth inhibition and dramatically reduced overall systemic toxicity compared to free carfilzomib. Finally, liposomal carfilzomib demonstrated enhanced synergy in combination with doxorubicin. Taken together, this study establishes the successful synthesis of liposomal carfilzomib nanoparticles that demonstrates improved therapeutic index and the potential to improve patient outcome in MM.
Assuntos
Antineoplásicos/farmacologia , Mieloma Múltiplo/tratamento farmacológico , Oligopeptídeos/farmacologia , Inibidores de Proteases/farmacologia , Animais , Antibióticos Antineoplásicos/farmacologia , Antineoplásicos/administração & dosagem , Linhagem Celular Tumoral , Doxorrubicina/farmacologia , Sinergismo Farmacológico , Humanos , Integrina alfa4beta1/efeitos dos fármacos , Integrinas/biossíntese , Lipossomos/química , Camundongos , Camundongos SCID , Nanopartículas , Oligopeptídeos/administração & dosagem , Tamanho da Partícula , Inibidores de Proteases/administração & dosagem , Solubilidade , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
In a previous study, we demonstrated a non-chromatographic affinity-based precipitation method, using trivalent haptens, for the purification of mAbs. In this study, we significantly improved this process by using a simplified bivalent peptidic hapten (BPH) design, which enables facile and rapid purification of mAbs while overcoming the limitations of the previous trivalent design. The improved affinity-based precipitation method (ABP(BPH)) combines the simplicity of salt-induced precipitation with the selectivity of affinity chromatography for the purification of mAbs. The ABP(BPH) method involves 3 steps: (i) precipitation and separation of protein contaminants larger than immunoglobulins with ammonium sulfate; (ii) selective precipitation of the target-antibody via BPH by inducing antibody-complex formation; (iii) solubilization of the antibody pellet and removal of BPH with membrane filtration resulting in the pure antibody. The ABP(BPH) method was evaluated by purifying the pharmaceutical antibody trastuzumab from common contaminants including CHO cell conditioned media, DNA, ascites fluid, other antibodies, and denatured antibody with >85% yield and >97% purity. Importantly, the purified antibody demonstrated native binding activity to cell lines expressing the target protein, HER2. Combined, the ABP(BPH) method is a rapid and scalable process for the purification of antibodies with the potential to improve product quality while decreasing purification costs.
Assuntos
Anticorpos Monoclonais/isolamento & purificação , Haptenos/imunologia , Sulfato de Amônio/química , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais Humanizados/química , Anticorpos Monoclonais Humanizados/imunologia , Anticorpos Monoclonais Humanizados/isolamento & purificação , Células CHO , Linhagem Celular , Precipitação Química , Cromatografia de Afinidade/métodos , Cromatografia de Fase Reversa/métodos , Cricetinae , Cricetulus , Filtração/métodos , Haptenos/química , Humanos , Peptídeos/química , Peptídeos/imunologia , Desnaturação Proteica , TrastuzumabRESUMO
Described here is a UV photo-cross-linking method that utilizes the NBS (nucleotide binding site) for site-specific covalent functionalization of antibodies with reactive thiol moieties (UV-NBS(Thiol)), while preserving antibody activity. By synthesizing an indole-3-butyric acid (IBA) conjugated version of cysteine we site-specifically photo-cross-linked a reactive thiol moiety to antibodies at the NBS. This thiol moiety can then be used as an orthogonally reactive location to conjugate various types of functional ligands that possess a thiol reactive group through disulfide bond formation or reaction with a maleimide functionalized ligand. Our results demonstrate the utility of the UV-NBS(Thiol) method by successfully functionalizing a prostate specific antigen antibody (IgG(PSA)) with IBA-Thiol and subsequent reaction with maleimide-fluorescein. An optimal UV energy of 0.5-1.5 J/cm(2) was determined to yield the most efficient photo-cross-linking and resulted in 1-1.5 conjugations per antibody while preserving antibody/antigen binding activity and Fc recognition. Utilizing the IBA-Thiol ligand allows for an efficient means of site-specifically conjugating UV sensitive functionalities to antibody NBS that would otherwise not have been amenable by the previously described UV-NBS photo-cross-linking method. The UV-NBS(Thiol) conjugation strategy can be utilized in various diagnostic and therapeutic applications with nearly limitless potential for the preparation of site-specific covalent conjugation of affinity tags, fluorescent molecules, peptides, and chemotherapeutics to antibodies.