RESUMEN
Rational design and robust formulation processes are critical for optimal delivery of mRNA by lipid nanoparticles (LNPs). Varying degrees of heterogeneity in mRNA-LNPs can affect their biophysical and functional properties. Given the profound complexity of mRNA-LNPs, it is critical to develop comprehensive and orthogonal analytical techniques for a better understanding of these formulations. To this end, we developed a robust ultracentrifugation method for density-based separation of subpopulations of mRNA-LNPs. Four LNP formulations encapsulating human erythropoietin (hEPO) with varying functionalities were synthesized using two ionizable lipids, A and B, and two helper lipids, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and 1,2-dierucoyl-sn-glycero-3-phosphoethanolamine (DEPE), along with cholesterol and DMG-PEG-2K. Upon ultracentrifugation on a sucrose gradient, a distinct pattern of "fractions" was observed across the gradient, from the less dense topmost fraction to the increasingly denser bottom fractions, which were harvested for comprehensive analyses. Parent LNPs, A-DOPE and B-DOPE, were resolved into three density-based fractions, each differing significantly in the hEPO expression following intravenous and intramuscular routes of administration. Parent B-DEPE LNPs resolved into two density-based fractions, with most of the payload and lipid content being attributed to the topmost fraction compared to the lower one, indicating some degree of heterogeneity, while parent A-DEPE LNPs showed remarkable homogeneity, as indicated by comparable in vivo potency, lipid numbers, and particle count among the three density-based fractions. This study is the first to demonstrate the application of density gradient-based ultracentrifugation (DGC) for a head-to-head comparison of heterogeneity as a function of biological performance and biophysical characteristics of parent mRNA-LNPs and their subpopulations.
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Lípidos , Nanopartículas , Humanos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Liposomas , Nanopartículas/metabolismo , ARN Interferente Pequeño/genéticaRESUMEN
One of the promises of nanoparticle (NP) carriers is the reformulation of promising therapeutics that have failed clinical development due to pharmacologic challenges. However, current nanomedicine research has been focused on the delivery of established and novel therapeutics. Here we demonstrate proof of the principle of using NPs to revive the clinical potential of abandoned compounds using wortmannin (Wtmn) as a model drug. Wtmn is a potent inhibitor of phosphatidylinositol 3' kinase-related kinases but failed clinical translation due to drug-delivery challenges. We engineered a NP formulation of Wtmn and demonstrated that NP Wtmn has higher solubility and lower toxicity compared with Wtmn. To establish the clinical translation potential of NP Wtmn, we evaluated the therapeutic as a radiosensitizer in vitro and in vivo. NP Wtmn was found to be a potent radiosensitizer and was significantly more effective than the commonly used radiosensitizer cisplatin in vitro in three cancer cell lines. The mechanism of action of NP Wtmn radiosensitization was found to be through the inhibition of DNA-dependent protein kinase phosphorylation. Finally, NP Wtmn was shown to be an effective radiosensitizer in vivo using two murine xenograft models of cancer. Our results demonstrate that NP drug-delivery systems can promote the readoption of abandoned drugs such as Wtmn by overcoming drug-delivery challenges.
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Androstadienos/farmacocinética , Sistemas de Liberación de Medicamentos/métodos , Nanopartículas , Neoplasias/terapia , Inhibidores de Proteínas Quinasas/farmacocinética , Fármacos Sensibilizantes a Radiaciones/farmacocinética , Androstadienos/toxicidad , Animales , Supervivencia Celular/efectos de los fármacos , Quimioradioterapia/métodos , Células HT29 , Humanos , Células KB , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones SCID , Fosforilación/efectos de los fármacos , Inhibidores de Proteínas Quinasas/toxicidad , Proteínas Proto-Oncogénicas c-akt/metabolismo , Fármacos Sensibilizantes a Radiaciones/toxicidad , Wortmanina , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Pulmonary delivery of mRNA via inhalation is a very attractive approach for RNA-based therapy for treatment of lung diseases. In this work, we have demonstrated successful development of an mRNA-lipid nanoparticle (LNP) dry powder product (DPP), wherein the LNPs were spray dried using hydroalcoholic solvent along with mannitol and leucine as excipients. The desired critical attributes for the DPP were accomplished by varying the excipients, lipid composition, concentration of LNPs, and weight percentage of mRNA. Leucine alone or in combination with mannitol improved the formulation by increasing the mRNA yield as well as decreasing the particle size. Intratracheal administration of the DPP in mice resulted in luciferase expression in the trachea and lungs indicating successful delivery of functional mRNA. Our results show formulation optimization of mRNA LNPs administered in the form of DPP results in an efficacious functional delivery with great promise for future development of mRNA therapeutics for lung diseases.
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Enfermedades Pulmonares , Nanopartículas , Ratones , Animales , Polvos/metabolismo , ARN Mensajero , Excipientes , Leucina , Pulmón/metabolismo , Manitol , Enfermedades Pulmonares/tratamiento farmacológico , Tamaño de la PartículaRESUMEN
Lipid nanoparticles (LNPs) have shown great promise as delivery vehicles to transport messenger ribonucleic acid (mRNA) into cells and act as vaccines for infectious diseases including COVID-19 and influenza. The ionizable lipid incorporated within the LNP is known to be one of the main driving factors for potency and tolerability. Herein, we describe a novel family of ionizable lipids synthesized with a piperazine core derived from the HEPES Good buffer. These ionizable lipids have unique asymmetric tails and two dissimilar degradable moieties incorporated within the structure. Lipids tails of varying lengths, degrees of unsaturation, branching, and the inclusion of additional ester moieties were evaluated for protein expression. We observed several key lipid structure activity relationships that correlated with improved protein production in vivo, including lipid tails of 12 carbons on the ester side and the effect of carbon spacing on the disulfide arm of the lipids. Differences in LNP physical characteristics were observed for lipids containing an extra ester moiety. The LNP structure and lipid bilayer packing, visualized through Cryo-TEM, affected the amount of protein produced in vivo. In non-human primates, the Good HEPES LNPs formulated with an mRNA encoding an influenza hemagglutinin (HA) antigen successfully generated functional HA inhibition (HAI) antibody titers comparable to the industry standards MC3 and SM-102 LNPs, demonstrating their promise as a potential vaccine.
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COVID-19 , Vacunas contra la Influenza , Gripe Humana , Animales , Humanos , HEPES , Membrana Dobles de Lípidos , Carbono , Ésteres , Vacunas de ARNmRESUMEN
The emergence of SARS-CoV-2 variants, especially Beta and Delta, has raised concerns about the reduced protection from previous infection or vaccination based on the original Wuhan-Hu-1 (D614) virus. To identify promising regimens for inducing neutralizing titers towards new variants, we evaluated monovalent and bivalent mRNA vaccines either as primary vaccination or as a booster in nonhuman primates (NHPs). Two mRNA vaccines, D614-based MRT5500 and Beta-based MRT5500ß, tested in sequential regimens or as a bivalent combination in naïve NHPs produced modest neutralizing titers to heterologous variants. However, when mRNA vaccines were administered as a booster to pre-immune NHPs, we observed a robust increase in neutralizing titers with expanded breadth towards all tested variants, and notably SARS-CoV-1. The breadth of the neutralizing response was independent of vaccine sequence or modality, as we further showed either MRT5500 or recombinant subunit Spike protein (with adjuvant) can serve as boosters to induce broadly neutralizing antibodies in the NHPs primed with MRT5500. The data support the notion that a third vaccination is key to boosting existing titers and improving the breadth of antibodies to address variants of concern, including those with an E484K mutation in the Receptor Binding Domain (RBD) (Beta, Gamma).
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COVID-19 , SARS-CoV-2 , Animales , Anticuerpos Neutralizantes , Anticuerpos Antivirales , Vacunas contra la COVID-19 , Humanos , Primates , Glicoproteína de la Espiga del Coronavirus , VacunaciónRESUMEN
Emergency use authorization of COVID vaccines has brought hope to mitigate pandemic of coronavirus disease 2019 (COVID-19). However, there remains a need for additional effective vaccines to meet the global demand and address the potential new viral variants. mRNA technologies offer an expeditious path alternative to traditional vaccine approaches. Here we describe the efforts to utilize an mRNA platform for rational design and evaluations of mRNA vaccine candidates based on the spike (S) glycoprotein of SARS-CoV-2. Several mRNA constructs of S-protein, including wild type, a pre-fusion stabilized mutant (2P), a furin cleavage-site mutant (GSAS) and a double mutant form (2P/GSAS), as well as others, were tested in animal models for their capacity to elicit neutralizing antibodies (nAbs). The lead 2P/GSAS candidate was further assessed in dose-ranging studies in mice and Cynomolgus macaques, and for efficacy in a Syrian golden hamster model. The selected 2P/GSAS vaccine formulation, designated MRT5500, elicited potent nAbs as measured in neutralization assays in all three preclinical models and more importantly, protected against SARS-CoV-2-induced weight loss and lung pathology in hamsters. In addition, MRT5500 elicited TH1-biased responses in both mouse and non-human primate (NHP), thus alleviating a hypothetical concern of potential vaccine-associated enhanced respiratory diseases known associated with TH2-biased responses. These data position MRT5500 as a viable vaccine candidate for entering clinical development.
RESUMEN
Recent approval of mRNA vaccines for emergency use against COVID-19 is likely to promote rapid development of mRNA-based vaccines targeting a wide range of infectious diseases. Compared to conventional approaches, this vaccine modality promises comparable potency while substantially accelerating the pace of development and deployment of vaccine doses. Already demonstrated successfully for single antigen vaccines such as for COVID-19, this technology could be optimized for complex multi-antigen vaccines. Herein, utilizing multiple influenza antigens, we demonstrated the suitability of the mRNA therapeutic (MRT) platform for such applications. Seasonal influenza vaccines have three or four hemagglutinin (HA) antigens of different viral subtypes. In addition, influenza neuraminidase (NA), a tetrameric membrane protein, is identified as an antigen that has been linked to protective immunity against severe viral disease. We detail the efforts in optimizing formulations of influenza candidates that use unmodified mRNA encoding full-length HA or full-length NA encapsulated in lipid nanoparticles (LNPs). HA and NA mRNA-LNP formulations, either as monovalent or as multivalent vaccines, induced strong functional antibody and cellular responses in non-human primates and such antigen-specific antibody responses were associated with protective efficacy against viral challenge in mice.
RESUMEN
The effects of nanoparticle (NP) properties, such as size, shape and surface charge, on their efficacy and toxicity have been studied extensively. However, the effect of controlled drug release on NP efficacy and toxicity has not been thoroughly evaluated in vivo. Our study aims to fill this knowledge gap. A key challenge in characterizing the relationship between drug release and therapeutic ratio is to fabricate NPs that differ only in their drug release profile but are otherwise identical. To overcome this challenge, we developed crosslinkable lipid shell (CLS) NPs, where the drug release kinetics can be modulated without changing any other NP property. Using CLS NPs with wortmannin and docetaxel as model drugs, we determined the relationship between the release kinetics and therapeutic efficacy and toxicity of the drugs. We have determined that drug release kinetics can affect the therapeutic efficacy of NP docetaxel and NP wortmannin in vitro and in vivo. Our study also demonstrates that a decrease in drug release kinetics can result in a decrease in the hepatotoxicity of CLS NP wortmannin. Using two model drugs, the current findings provide the first direct evidence that NP drug release profile is a critical factor in determining the NP therapeutics' efficacy and toxicity in vivo.
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Androstadienos , Antineoplásicos , Inmunosupresores , Nanopartículas/química , Neoplasias Experimentales/tratamiento farmacológico , Taxoides , Androstadienos/química , Androstadienos/farmacocinética , Androstadienos/farmacología , Animales , Antineoplásicos/química , Antineoplásicos/farmacocinética , Antineoplásicos/farmacología , Preparaciones de Acción Retardada/química , Preparaciones de Acción Retardada/farmacocinética , Preparaciones de Acción Retardada/farmacología , Docetaxel , Humanos , Inmunosupresores/química , Inmunosupresores/farmacocinética , Inmunosupresores/farmacología , Cinética , Masculino , Ratones , Ratones Desnudos , Neoplasias Experimentales/patología , Taxoides/química , Taxoides/farmacocinética , Taxoides/farmacología , WortmaninaRESUMEN
pH-triggered lipid-membranes designed from biophysical principles are evaluated in the form of targeted liposomal doxorubicin with the aim to ultimately better control the growth of vascularized tumors. We compare the antitumor efficacy of anti-HER2/neu pH-triggered lipid vesicles encapsulating doxorubicin to the anti-HER2/neu form of an FDA approved liposomal doxorubicin of DSPC/cholesterol-based vesicles. The HER2/neu receptor is chosen due to its abundance in human breast cancers and its connection to low prognosis. On a subcutaneous murine BT474 xenograft model, superior control of tumor growth is demonstrated by targeted pH-triggered vesicles relative to targeted DSPC/cholesterol-based vesicles (35% vs. 19% decrease in tumor volume after 32 days upon initiation of treatment). Superior tumor control is also confirmed on SKBR3 subcutaneous xenografts of lower HER2/neu expression. The non-targeted form of pH-triggered vesicles encapsulating doxorubicin results also in better tumor control relative to the non-targeted DSPC/cholesterol-based vesicles (34% vs. 41% increase in tumor volume). Studies in BT474 multicellular spheroids suggest that the observed efficacy could be attributed to release of doxorubicin directly into the acidic tumor interstitium from pH-triggered vesicles extravasated into the tumor but not internalized by cancer cells. pH-triggered liposome carriers engineered from gel-phase bilayers that reversibly phase-separate with lowering pH, form transiently defective interfacial boundaries resulting in fast release of encapsulated doxorubicin. Our studies show that pH-triggered liposomes release encapsulated doxorubicin intracellularly and intratumorally, and may improve tumor control at the same or even lower administered doses relative to FDA approved liposomal chemotherapy.
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Antineoplásicos/farmacología , Doxorrubicina/farmacología , Sistemas de Liberación de Medicamentos/métodos , Espacio Intracelular/efectos de los fármacos , Animales , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Femenino , Humanos , Liposomas/química , Ratones , Ratones Desnudos , Esferoides Celulares/efectos de los fármacos , Esferoides Celulares/patología , Factores de Tiempo , Resultado del Tratamiento , Carga Tumoral/efectos de los fármacosRESUMEN
Peritoneal metastasis is a major cause of morbidity and mortality in ovarian cancer. While intraperitoneal chemotherapy and radiotherapy have shown favorable clinical results, both are limited by their non-targeted nature. We aimed to develop a biologically targeted nanoparticle therapeutic for the treatment of ovarian cancer peritoneal metastasis. Folate-targeted nanoparticles encapsulating chemotherapy and/or radiotherapy were engineered. Paclitaxel (Ptxl) was used as the chemotherapeutic and yittrium-90 ((90)Y) was employed as the therapeutic radioisotope. Folate was utilized as the targeting ligand as most ovarian cancers overexpress the folate receptor. Nanoparticle characterization studies showed monodispersed particles with controlled Ptxl release. Folate targeting ligand mediated the uptake of NPs into tumor cells. In vitro efficacy studies demonstrated folate-targeted NPs containing chemoradiotherapy was the most effective therapeutic compared to folate-targeted NPs containing a single therapeutic or any non-targeted NP therapeutics. In vivo efficacy studies using an ovarian peritoneal metastasis model showed that folate-targeted NP therapeutics were significantly more effective than non-targeted NP therapeutics. Among the folate-targeted therapeutics, the NP containing chemoradiotherapy appeared to be the most effective. Our results suggest that folate-targeted nanoparticles containing chemoradiotherapy have the potential as a treatment for ovarian peritoneal metastasis.
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Antineoplásicos Fitogénicos/uso terapéutico , Ácido Fólico/metabolismo , Nanopartículas , Neoplasias Ováricas/patología , Paclitaxel/uso terapéutico , Neoplasias Peritoneales/secundario , Neoplasias Peritoneales/terapia , Animales , Antineoplásicos Fitogénicos/administración & dosificación , Antineoplásicos Fitogénicos/metabolismo , Western Blotting , Terapia Combinada , Femenino , Humanos , Ratones , Ratones Desnudos , Microscopía Fluorescente , Paclitaxel/administración & dosificación , Paclitaxel/metabolismo , Neoplasias Peritoneales/tratamiento farmacológico , Neoplasias Peritoneales/radioterapiaRESUMEN
Nanotechnology is a relatively new branch of science that involves harnessing the unique properties of particles that are nanometers in scale (nanoparticles). Nanoparticles can be engineered in a precise fashion where their size, composition and surface chemistry can be carefully controlled. This enables unprecedented freedom to modify some of the fundamental properties of their cargo, such as solubility, diffusivity, biodistribution, release characteristics and immunogenicity. Since their inception, nanoparticles have been utilized in many areas of science and medicine, including drug delivery, imaging, and cell biology(1-4). However, it has not been fully utilized outside of "nanotechnology laboratories" due to perceived technical barrier. In this article, we describe a simple method to synthesize a polymer based nanoparticle platform that has a wide range of potential applications. The first step is to synthesize a diblock co-polymer that has both a hydrophobic domain and hydrophilic domain. Using PLGA and PEG as model polymers, we described a conjugation reaction using EDC/NHS chemistry(5) (Fig 1). We also discuss the polymer purification process. The synthesized diblock co-polymer can self-assemble into nanoparticles in the nanoprecipitation process through hydrophobic-hydrophilic interactions. The described polymer nanoparticle is very versatile. The hydrophobic core of the nanoparticle can be utilized to carry poorly soluble drugs for drug delivery experiments6. Furthermore, the nanoparticles can overcome the problem of toxic solvents for poorly soluble molecular biology reagents, such as wortmannin, which requires a solvent like DMSO. However, DMSO can be toxic to cells and interfere with the experiment. These poorly soluble drugs and reagents can be effectively delivered using polymer nanoparticles with minimal toxicity. Polymer nanoparticles can also be loaded with fluorescent dye and utilized for intracellular trafficking studies. Lastly, these polymer nanoparticles can be conjugated to targeting ligands through surface PEG. Such targeted nanoparticles can be utilized to label specific epitopes on or in cells(7-10).
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Nanopartículas/química , Nanotecnología/métodos , Polímeros/química , Interacciones Hidrofóbicas e Hidrofílicas , Ácido Láctico/química , Polietilenglicoles/química , Poliglactina 910/química , Ácido Poliglicólico/química , Copolímero de Ácido Poliláctico-Ácido PoliglicólicoRESUMEN
Nanoparticle (NP) chemotherapeutics hold great potential as radiosensitizers. Their unique properties, such as preferential accumulation in tumors and their ability to target tumors through molecular targeting ligands, are ideally suited for radiosensitization. We aimed to develop a molecularly targeted nanoparticle formulation of docetaxel (Dtxl) and evaluate its property as a radiosensitizer. Using a biodegradable and biocompatible lipid-polymer NP platform and folate as a molecular targeting ligand, we engineered a folate-targeted nanoparticle (FT-NP) formulation of Dtxl. These NPs have sizes of 72 ± 4 nm and surface charges of -42 ± 8 mV. Using folate receptor overexpressing KB cells and folate receptor low HTB-43 cells, we showed folate-mediated intracellular uptake of NPs. In vitro radiosensitization studies initially showed FT-NP is less effective than Dtxl as a radiosensitizer. However, the radiosensitization efficacy is dependent on the timing of radiotherapy. In vitro radiosensitization conducted with irradiation given at the optimal time (24 h) showed FT-NP Dtxl is as effective as Dtxl. When FT-NP Dtxl is compared to Dtxl and nontargeted nanoparticle (NT-NP) Dtxl in vivo, FT-NP was found to be significantly more effective than Dtxl or NT-NP Dtxl as a radiosensitizer. We also confirmed that radiosensitization is dependent on timing of irradiation in vivo. In summary, FT-NP Dtxl is an effective radiosensitizer in folate-receptor overexpressing tumor cells. Time of irradiation is critical in achieving maximal efficacy with this nanoparticle platform. To the best of our knowledge, our report is the first to demonstrate the potential of molecularly targeted NPs as a promising new class of radiosensitizers.
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Ácido Fólico/metabolismo , Neoplasias de Cabeza y Cuello/radioterapia , Terapia Molecular Dirigida/métodos , Nanopartículas/química , Polímeros/química , Fármacos Sensibilizantes a Radiaciones/química , Taxoides/química , Animales , Transporte Biológico , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/efectos de la radiación , Química Farmacéutica , Docetaxel , Transportadores de Ácido Fólico/deficiencia , Transportadores de Ácido Fólico/metabolismo , Neoplasias de Cabeza y Cuello/tratamiento farmacológico , Neoplasias de Cabeza y Cuello/metabolismo , Humanos , Células KB , Ratones , Fármacos Sensibilizantes a Radiaciones/farmacología , Fármacos Sensibilizantes a Radiaciones/uso terapéutico , Taxoides/farmacología , Taxoides/uso terapéutico , Factores de Tiempo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
To enable selective cell-kill, we designed functionalized lipid vesicles with pH-triggered heterogeneous membranes and encapsulated doxorubicin that exhibit tunable surface topography. These vesicles "hide" (mask) the targeting ligands from their surface during circulation in the blood, and only progressively "expose" these ligands as they gradually penetrate deeper into the tumor interstitium, where after endocytosis they burst release their contents. The stimulus to activate the binding reactivity is the pH gradient between the blood stream (pH 7.4-7.0) and the increasingly acidic pH inside the tumor interstitium (pH 6.7-6.5). Doxorubicin release is activated at the endosomal pH 5.5-5.0. We show that tunable functionalized vesicles exhibit environmentally-dependent (pH-dependent) association with cancer cells resulting in high cell-kill selectivity. When lowering the extracellular pH from 7.4 to 6.5, tunable functionalized vesicles deliver doxorubicin to cancer cells that increases from 41% to 93% of maximum resulting in cancer cell killing that increases from 23 to 71% of maximum, respectively. This proof-of-concept shows the potential of tunable targeted liposomal chemotherapy to selectively kill cancer cells in an environmentally-dependent way.
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Antibióticos Antineoplásicos , Línea Celular Tumoral/efectos de los fármacos , Doxorrubicina , Sistemas de Liberación de Medicamentos/métodos , Lípidos/química , Antibióticos Antineoplásicos/administración & dosificación , Antibióticos Antineoplásicos/química , Antibióticos Antineoplásicos/farmacología , Rastreo Diferencial de Calorimetría , Doxorrubicina/administración & dosificación , Doxorrubicina/química , Doxorrubicina/farmacología , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Concentración de Iones de Hidrógeno , Ensayo de Materiales , Modelos MolecularesRESUMEN
During endocytosis, pH-triggered release of encapsulated therapeutics from delivery carriers may accelerate their intracellular trafficking increasing therapeutic efficacy. To improve the therapeutic potential of targeted immunochemotherapy using anti-HER2/neu liposomal doxorubicin, we exploit the formation of leaky heterogeneities on rigid lipid bilayers to extensively release doxorubicin during endocytosis. We have previously demonstrated that pH-dependent formation of phase-separated lipid heterogeneities on the plane of a bilayer membrane increases the permeability of bilayers when they are composed of lipid pairs with rigid non-matching acyl chain lengths. This was suggested to be due to defective packing among lipids residing at the interfaces of lipid domains. Here we design nanometer-size antiHER2/neu-labeled PEGylated vesicles composed of lipid pairs with longer non-matching acyl chain lengths (n=18 and 21). These vesicles exhibit superior killing efficacy of cancer cells compared to established liposome formulations, and their killing efficacy is similar to the effect of combined free doxorubicin and free antiHER2/neu antibody. Other transport-related properties such as liposome blood circulation times, and specific binding and internalization by cancer cells are unaffected. These results demonstrate the potential of vesicles with pH-triggered leaky heterogeneities to increase the therapeutic potential of targeted immunochemotherapy.
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Quimioterapia/métodos , Inmunoterapia/métodos , Membrana Dobles de Lípidos , Liposomas/química , Animales , Línea Celular Tumoral , Membrana Celular/metabolismo , Terapia Combinada/métodos , Doxorrubicina/administración & dosificación , Sistemas de Liberación de Medicamentos , Ensayos de Selección de Medicamentos Antitumorales , Endocitosis , Femenino , Humanos , Concentración de Iones de Hidrógeno , Lípidos/química , Ratones , Ratones Endogámicos BALB C , Polietilenglicoles/químicaRESUMEN
During direct cell-to-cell communication, lipids on the extracellular side of plasma membranes reorganize, and membrane-associated communication-related molecules colocalize. At colocalization sites, sometimes identified as rafts, the local cell surface topography and reactivity are altered. The processes regulating these changes are largely unknown. On model lipid membranes, study of simplified processes that control surface topography and reactivity may potentially contribute to the understanding and control of related cell functions and associated diseases. Integration of these processes on nanometer-sized lipid vesicles used as drug delivery carriers would precisely control their interactions with diseased cells minimizing toxicities. Here we design such basic pH-dependent processes on model functionalized lipid bilayers, and we demonstrate reversible sharp changes in binding reactivity within a narrow pH window. Cholesterol enables tuning of the membrane reorganization to occur at pH values not necessarily close to the reported pK(a)'s of the constituent titratable lipids, and bilayer reorganization over repeated cycles of induced pH changes exhibits hysteresis.
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Membrana Dobles de Lípidos/química , Rastreo Diferencial de Calorimetría , Colesterol/química , Concentración de Iones de Hidrógeno , Modelos QuímicosRESUMEN
Heterogeneous lipid membranes tuned by pH were evaluated at 37 degrees C in the form of PEGylated vesicles composed of lipid pairs with dipalmitoyl ( n = 16) and distearoyl ( n = 18) chain lengths. One lipid type was chosen to have the titratable moiety phosphatidic acid on its headgroup, and the other lipid type was chosen to have a phosphatidylcholine headgroup. The effect of pH on the formation of lipid heterogeneities and on membrane permeability was studied on vesicles composed of lipid pairs with matching and nonmatching chain lengths. The formation of lipid heterogeneities increases with decreasing pH in membranes composed of lipid pairs with either matching or nonmatching chain lengths. Increased permeability with decreasing pH was exhibited only by membranes composed of lipid pairs with nonmatching chain lengths. Permeability rates correlate strongly with the predicted extent of interfacial boundaries of heterogeneities, suggesting defective packing among nonmatching acyl chains of lipids. In heterogeneous mixtures with one lipid type in the fluid state ( n = 12), the dependence of membrane permeability on pH is weaker. In the presence of serum proteins, PEGylated gel-phase vesicles containing lipid pairs with nonmatching chain lengths exhibit faster release rates with decreasing pH compared to measured release rates in phosphate buffer, suggesting a second mechanism of formation of separated phases. PEGylated vesicles composed of lipid pairs with nonmatching chain lengths labeled with internalizing anti-HER2/neu antibodies that target overexpressed antigens on the surface of SKOV3-NMP2 ovarian cancer cells exhibit specific cancer cell targeting, followed by extensive internalization (more than 84% of bound vesicles) and fast release of contents intracellularly. These PEGylated vesicles composed of rigid membranes for long blood circulation times that exhibit pH-dependent release of contents intracellularly could become potent drug delivery carriers for the targeted therapy of solid tumors.