RESUMO
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
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
In this study, we describe the development of liposomal bortezomib nanoparticles, which was accomplished by synthesizing bortezomib prodrugs with reversible boronic ester bonds and then incorporating the resulting prodrugs into the nanoparticles via surface conjugation. Initially, several prodrug candidates were screened based upon boronic ester stability using isobutylboronic acid as a model boronic acid compound. The two most stable candidates were then selected to create surface conjugated bortezomib prodrugs on the liposomes. Our strategy yielded stable liposomal bortezomib nanoparticles with a narrow size range of 100 nm and with high reproducibility. These liposomal bortezomib nanoparticles demonstrated significant proteasome inhibition and cytotoxicity against multiple myeloma cell lines in vitro and remarkable tumor growth inhibition with reduced systemic toxicity compared to free bortezomib in vivo. Taken together, this study demonstrates the incorporation of bortezomib into liposomal nanoparticles via reversible boronic ester bond formation to enhance the therapeutic index for improved patient outcome.
Assuntos
Antineoplásicos/administração & dosagem , Ácidos Borônicos/administração & dosagem , Pró-Fármacos/administração & dosagem , Pirazinas/administração & dosagem , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Apoptose , Ácidos Borônicos/síntese química , Ácidos Borônicos/química , Bortezomib , Linhagem Celular Tumoral , Ésteres , Humanos , Lipossomos , Camundongos SCID , Mieloma Múltiplo/tratamento farmacológico , Nanopartículas , Transplante de Neoplasias , Pró-Fármacos/síntese química , Pró-Fármacos/química , Complexo de Endopeptidases do Proteassoma/metabolismo , Pirazinas/síntese química , Pirazinas/químicaRESUMO
Cisplatin has long been the first line of treatment for a variety of solid tumors. However, poor pharmacokinetics and high incidences of resistance in the clinic have motivated the production of numerous alternative Pt-based anticancer species. Recently, photosensitive Pt(IV) complexes have garnered much interest because they offer a method of selective induction of active Pt(II) at the tumor site by UVA irradiation. Here, we report the first synthesis, in vitro and in vivo characterization of a novel series of photosensitive Pt(IV)azide prodrugs and micellar nanoparticle formulations thereof. Upon mild UVA irradiation, both free Pt(IV) complexes and micellar nanoparticles rapidly released biologically active Pt(II), capable of binding to 5'-GMP,while remaining extremely stable in the dark. In vitro, uptake of photosensitive Pt(IV) prodrugs by ovarian cancer SKOV-3 cells was greatly enhanced with the micellar nanoparticles compared to their free prodrug analogs, as well as cisplatin and oxaliplatin. Increased cytotoxicity was observed upon UVA treatment, with up to a 13-fold enhancement over oxaliplatin for the micellar nanoparticles. In vivo bioavailability of micellar nanoparticles was enhanced ~10 fold over free Pt(IV) prodrugs. Importantly, micellar nanoparticles demonstrated significantly improved efficacy against H22 murine hepatocarcinoma, showing decreased systemic toxicity and increased tumor growth inhibition relative to small molecule drugs. These findings establish that photosensitive Pt(IV) complexes, specifically when formulated into micellar nanoparticles, have the potential to offer a robust platform for the controlled delivery and selective activation of Pt-based anticancer therapeutics.
Assuntos
Antineoplásicos/administração & dosagem , Azidas/administração & dosagem , Preparações de Ação Retardada/química , Nanopartículas/química , Compostos Organoplatínicos/administração & dosagem , Pró-Fármacos/administração & dosagem , Animais , Antineoplásicos/química , Antineoplásicos/uso terapêutico , Azidas/uso terapêutico , Linhagem Celular Tumoral , Humanos , Fígado/efeitos dos fármacos , Fígado/patologia , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/patologia , Camundongos , Compostos Organoplatínicos/química , Compostos Organoplatínicos/uso terapêutico , Pró-Fármacos/química , Pró-Fármacos/uso terapêutico , Raios UltravioletaRESUMO
Nanomedicine, particularly liposomal drug delivery, has expanded considerably over the past few decades, and several liposomal drugs are already providing improved clinical outcomes. Liposomes have now progressed beyond simple, inert drug carriers and can be designed to be highly responsive in vivo, with active targeting, increased stealth, and controlled drug-release properties. Ligand-targeted liposomes (LTLs) have the potential to revolutionize the treatment of cancer. However, these highly engineered liposomes generate new problems, such as accelerated clearance from circulation, compromised targeting owing to non-specific serum protein binding, and hindered tumor penetration. This article highlights recent challenges facing LTL strategies and describes the advanced design elements used to circumvent them.
Assuntos
Biotecnologia , Sistemas de Liberação de Medicamentos , Lipossomos , Nanomedicina , Animais , Antineoplásicos , Linhagem Celular Tumoral , Humanos , Ligantes , CamundongosRESUMO
Ligand-targeted nanoparticles are emerging drug delivery vehicles for cancer therapy. Here, we demonstrate that the cellular uptake of peptide-targeted liposomes and micelles can be significantly enhanced by increasing the hydrophilicity of the targeting peptide sequence while simultaneously optimizing the EG peptide-linker length. Two distinct disease models were analyzed, as the nanoparticles were functionalized with either VLA-4 or HER2 antagonistic peptides to target multiple myeloma or breast cancer cells, respectively. Our results demonstrated that including a short oligolysine chain adjacent to the targeting peptide sequence effectively increased cellular uptake of targeted nanoparticles up to â¼80-fold using an EG6 peptide-linker in liposomes and â¼27-fold using an EG18 peptide-linker in micelles for the VLA-4/multiple myeloma system. Similar trends were also observed in the HER2/breast cancer system with the EG18 peptide-linker resulting in optimal uptake for both types of nanoparticles. Cellular uptake efficiency of these formulations was also confirmed under fluidic conditions mimicking physiological systems. Taken together, these results demonstrated the significance of using the right design elements to improve the cellular uptake of nanoparticles.
Assuntos
Integrina alfa4beta1/metabolismo , Nanocápsulas/química , Neoplasias Experimentais/metabolismo , Peptídeos/química , Polietilenoglicóis/química , Receptor ErbB-2/metabolismo , Linhagem Celular Tumoral , Reagentes de Ligações Cruzadas/química , Humanos , Interações Hidrofóbicas e Hidrofílicas , Nanocápsulas/ultraestrutura , Tamanho da PartículaRESUMO
Liposome Extruder Purification (LEP) allows for the rapid purification of diverse liposome formulations using the same extrusion apparatus employed during liposome formation. The LEP process provides a means for purifying functionalized liposomes from non-conjugated drug or protein contaminants with >93% liposome recovery and >93% contaminant removal in a single step.
Assuntos
Fracionamento Químico/métodos , Lipossomos/isolamento & purificação , Animais , Bovinos , Química Farmacêutica , Lipossomos/química , Fatores de TempoRESUMO
Nanoparticles formed via electrostatic interactions between methoxy-polyethylene glycol-block-poly(glutamic acid) (MPEG-PGA) and a multinuclear platinum(II) drug, di-cisPt, exhibit efficient platinum loading, improved cellular uptake, in vitro cytotoxicity, and enhanced in vivo efficacy.
Assuntos
Antineoplásicos/administração & dosagem , Cisplatino/administração & dosagem , Portadores de Fármacos/administração & dosagem , Nanopartículas/administração & dosagem , Antineoplásicos/química , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cisplatino/química , Portadores de Fármacos/química , Humanos , Micelas , Nanopartículas/química , Polietilenoglicóis/química , Ácido Poliglutâmico/química , Eletricidade EstáticaRESUMO
The conserved nucleotide binding site (NBS), found in the Fab variable domain of all antibody isotypes, remains a not-so-widely known and under-utilized site. Here, we describe a UV photocrosslinking method (UV-NBS) that utilizes the NBS for site-specific covalent functionalization of antibodies, while preserving antibody activity. We identified a small molecule, indole-3-butyric acid (IBA), which has affinity for the NBS (K(d) = 1-8 µM) and can be photocrosslinked to antibodies upon UV energy exposure. By synthesizing their IBA conjugated versions, we have successfully photocrosslinked various types of functional ligands to antibodies at the NBS, including affinity tags (biotin), fluorescent molecules (FITC), peptides (iRGD), and chemotherapeutics (paclitaxel). An optimal UV exposure of 1-2 J/cm(2) yielded the most efficient photocrosslinking and resulted in 1-2 conjugations per antibody, while preserving the antigen binding activity and Fc related functions. Analysis of the photocrosslinked conjugates using western blotting, mass spectrometry, and computational docking simulations demonstrated that the photocrosslinking specifically takes place at the Y/F42 residue in framework region 2 of the antibody light chain. Taken together, the UV-NBS method provides a practical, site-specific, and chemically efficient method to functionalize antibodies with significant implications in diagnostic and therapeutic settings.
Assuntos
Anticorpos/metabolismo , Sequência Conservada , Reagentes de Ligações Cruzadas/metabolismo , Nucleotídeos/metabolismo , Raios Ultravioleta , Sequência de Aminoácidos , Animais , Anticorpos/química , Anticorpos Monoclonais Murinos/química , Anticorpos Monoclonais Murinos/metabolismo , Antígenos/metabolismo , Sítios de Ligação , Biotina/metabolismo , Western Blotting , Soluções Tampão , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Imunoglobulina G/química , Imunoglobulina G/metabolismo , Indóis/metabolismo , Ligantes , Espectrometria de Massas , Simulação de Acoplamento Molecular , Dados de Sequência Molecular , Oligopeptídeos/química , Oligopeptídeos/metabolismo , Paclitaxel/farmacologia , Ligação Proteica/efeitos da radiação , Receptores Fc/metabolismo , Rituximab , TermodinâmicaRESUMO
This Article describes an affinity-based precipitation method for the rapid and nonchromatographic purification of bivalently active monoclonal antibodies by combining the selectivity of affinity chromatography with the simplicity of salt-induced precipitation. This procedure involves (i) precipitation of proteins heavier than immunoglobulins with ammonium sulfate; (ii) formation and selective precipitation of cyclic antibody complexes created by binding to trivalent haptens specific for the antibody; and (iii) membrane filtration of the solubilized antibody pellet to remove the trivalent hapten from the purified antibody. We applied this technique to the purification of two pharmaceutical antibodies, trastuzumab and rituximab, by synthesizing trivalent haptens specific for each antibody. Using this method, we were able to purify both antibodies from typical contaminants including CHO cell conditioned media, ascites fluid, DNA, and other antibodies with yields >85% and with >95% purity. The purified antibodies displayed native binding levels to cell lines expressing the target proteins demonstrating that the affinity-based precipitation method did not adversely affect the antibodies. The selectivity of the affinity-based precipitation method for bivalently active antibodies was established by purifying trastuzumab from a solution containing both active and chemically denatured trastuzumab. Prior to purification, the solutions displayed 20-76% reduction in binding activity, and after purification, native binding activity was restored, indicating that the purified product contained only bivalently active antibody. Taken together, the affinity-based precipitation method provides a rapid and straightforward process for the purification of antibodies with the potential to improve product quality while decreasing the purification costs at both the lab and the industrial scale.
Assuntos
Anticorpos Monoclonais Humanizados/química , Anticorpos Monoclonais Humanizados/isolamento & purificação , Anticorpos Monoclonais Murinos/química , Anticorpos Monoclonais Murinos/isolamento & purificação , Ascite , Precipitação Química , Sulfato de Amônio/química , Animais , Anticorpos Monoclonais Humanizados/imunologia , Anticorpos Monoclonais Murinos/imunologia , Especificidade de Anticorpos , Células CHO , Cricetinae , Cricetulus , Meios de Cultivo Condicionados/química , DNA/isolamento & purificação , Haptenos/imunologia , Desnaturação Proteica , Rituximab , TrastuzumabRESUMO
PEGylated liposomes are attractive pharmaceutical nanocarriers; however, literature reports of ligand-targeted nanoparticles have not consistently shown successful results. Here, we employed a multifaceted synthetic strategy to prepare peptide-targeted liposomal nanoparticles with high purity, reproducibility, and precisely controlled stoichiometry of functionalities to evaluate the role of liposomal PEG coating, peptide EG-linker length, and peptide valency on cellular uptake in a systematic manner. We analyzed these parameters in two distinct disease models where the liposomes were functionalized with either HER2- or VLA-4-antagonistic peptides to target HER2-overexpressing breast cancer cells or VLA-4-overexpressing myeloma cells, respectively. When targeting peptides were tethered to nanoparticles with an EG45 (â¼PEG2000) linker in a manner similar to a more traditional formulation, their cellular uptake was not enhanced compared to non-targeted versions regardless of the liposomal PEG coating used. Conversely, reduction of the liposomal PEG to PEG350 and the peptide linker to EG12 dramatically enhanced cellular uptake by â¼9 fold and â¼100 fold in the breast cancer and multiple myeloma cells, respectively. Uptake efficiency reached a maximum and a plateau with â¼2% peptide density in both disease models. Taken together, these results demonstrate the significance of using the right design elements such as the appropriate peptide EG-linker length in coordination with the appropriate liposomal PEG coating and optimal ligand density in efficient cellular uptake of liposomal nanoparticles.