RESUMO
PURPOSE: To investigate the applicability of fusion biotoxins combining pore-forming toxins (PFTs) and ribosome-inactivating proteins (RIPs) for the anti-cancer treatment. METHODS: Membrane active PFTs tend to destabilize cell membranes of tumor cells, but lack a warhead inducing significant cause of cell death. Cell-impermeable RIPs possess a powerful warhead, yet not able to enter the tumor cells. To address these challenges for anti-tumor effects, we introduced a fusion strategy of conjugating melittin (a PFT) and gelonin (a type 1 RIP) via chemical and recombinant methods, followed by in vitro assays and in vivo animal studies. RESULTS: In vitro characterization results confirmed that the chimeric gelonin-melittin fusion proteins retained equivalent intrinsic activity to that of unmodified gelonin in inhibiting protein translation. However, chemically conjugated gelonin-melittin (cGel-Mel) and recombinant chimeric gelonin-melittin fusion (rGel-Mel) exhibited greater cell uptake, yielding a significantly enhanced cytotoxic activity over treatment of gelonin, melittin or physical mixture of gelonin and melittin. Remarkably, cGel-Mel and rGel-Mel displayed 32- and 10-fold lower IC50 than gelonin in the cell lines. The superior anti-tumor efficacy of multivalent cGel-Mel to monovalent rGel-Mel suggested that valency could be a crucial factor for the extent of melittin-mediated cell uptake. Tumoricidal effects observed from animal studies were in good accordance with our findings from the cellular assays. CONCLUSIONS: This study successfully demonstrated that fusion of biotoxins could provide a simple yet effective way to synergistically augment their anti-tumor activity.
Assuntos
Antineoplásicos Fitogênicos/química , Meliteno/química , Proteínas Recombinantes de Fusão/química , Proteínas Inativadoras de Ribossomos Tipo 1/química , Toxinas Biológicas/química , Animais , Antineoplásicos Fitogênicos/farmacologia , Linhagem Celular , Linhagem Celular Tumoral , Cães , Células HeLa , Humanos , Células Madin Darby de Rim Canino , Meliteno/farmacologia , Camundongos , Neoplasias/tratamento farmacológico , Ratos , Proteínas Recombinantes de Fusão/farmacologia , Proteínas Inativadoras de Ribossomos/química , Proteínas Inativadoras de Ribossomos/farmacologia , Proteínas Inativadoras de Ribossomos Tipo 1/farmacologia , Toxinas Biológicas/farmacologiaRESUMO
For the past 20 years, we have witnessed an unprecedented and, indeed, rather miraculous event of how cell-penetrating peptides (CPPs), the naturally originated penetrating enhancers, help overcome the membrane barrier that has hindered the access of bio-macromolecular compounds such as genes and proteins into cells, thereby denying their clinical potential to become potent anti-cancer drugs. By taking the advantage of the unique cell-translocation property of these short peptides, various payloads of proteins, nucleic acids, or even nanoparticle-based carriers were delivered into all cell types with unparalleled efficiency. However, non-specific CPP-mediated cell penetration into normal tissues can lead to widespread organ distribution of the payloads, thereby reducing the therapeutic efficacy of the drug and at the same time increasing the drug-induced toxic effects. In view of these challenges, we present herein a review of the new designs of CPP-linked vehicles and strategies to achieve highly effective yet less toxic chemotherapy in combating tumor oncology.
Assuntos
Antineoplásicos/administração & dosagem , Peptídeos Penetradores de Células/administração & dosagem , Portadores de Fármacos/administração & dosagem , Neoplasias/tratamento farmacológico , Animais , Antineoplásicos/farmacocinética , Peptídeos Penetradores de Células/farmacocinética , Portadores de Fármacos/farmacocinética , Endocitose , Humanos , Concentração de Íons de Hidrogênio , Permeabilidade , Distribuição TecidualRESUMO
Currently, oral administration of insulin still remains the best option to avoid the burden of repeated subcutaneous injections and to improve its pharmacokinetics. The objective of the present investigation was to demonstrate the absorption mechanism of insulin in the physical complexation of deoxycholyl-l-lysyl-methylester (DCK) for oral delivery. The oral insulin/DCK complex was prepared by making a physical complex of insulin aspart with DCK through ion-pair interaction in water. For the cellular uptake study, fluorescein-labeled insulin or DCK were prepared according to a standard protocol and applied to Caco-2 or MDCK cell lines. For the PK/PD studies, we performed intrajejunal administration of different formulation of insulin/DCK complex to diabetic rats. The resulting insulin and DCK complex demonstrated greatly enhanced lipophilicity as well as increased permeation across Caco-2 monolayers. The immunofluorescence study revealed the distribution of the complex in the cytoplasm of Caco-2 cells. Moreover, in the apical sodium bile acid transporter (ASBT) transfected MDCK, the insulin/DCK complex showed interaction with ASBT, and also demonstrated absorption through passive diffusion. We could not find that any evidence of endocytosis in relation to the uptake of insulin complex in vitro. In the rat intestine model, the highest absorption of insulin complex was observed in the jejunum at 1 h and then in the ileum at 2-4 h. In PK/PD study, the complex showed a similar PK profile to that of SC insulin. Overall, the study showed that the effect of DCK on enhancing the absorption of insulin resulted from transcellular processes as well as bile acid transporter activity.
Assuntos
Ácido Quenodesoxicólico/análogos & derivados , Portadores de Fármacos/química , Insulina/química , Intestino Delgado/metabolismo , Lisina/análogos & derivados , Administração Oral , Animais , Células CACO-2 , Ácido Quenodesoxicólico/química , Ácido Quenodesoxicólico/farmacocinética , Cães , Portadores de Fármacos/farmacocinética , Humanos , Insulina/farmacocinética , Jejuno/metabolismo , Lisina/química , Lisina/farmacocinética , Células Madin Darby de Rim Canino , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
PURPOSE: To investigate the feasibility of applying PTD-modified ATTEMPTS (Antibody Targeted Triggered Electrically Modified Prodrug-Type Strategy) for enhanced toxin therapy for the treatment of cancer. METHODS: A heparin-functionalized murine anti-CEA monoclonal antibody (mAb), T84.66-heparin (T84.66-Hep), was chemically synthesized and characterized for specific binding to CEA overexpressed cells. The T84.66-Hep was then applied to the PTD-modified ATTEMPTS approach and the crucial features of the drug delivery system (DDS), 'antibody targeting' and 'heparin/protamine-based prodrug', were evaluated in vitro to examine whether it could selective delivery a PTD-modified toxin, recombinant TAT-gelonin chimera (TAT-Gel), to CEA high expression cancer cells (LS174T). Furthermore, the feasibility of the drug delivery system (DDS) was assessed in vivo by biodistribution and efficacy studies using LS174T s.c. xenograft tumor bearing mice. RESULTS: T84.66-Hep displayed specific binding, but limited internalization (35% after 48 h incubation) to CEA high expression LS174T cells over low expression HCT116 cells. When mixed together with TAT-Gel, the T84.66-Hep formed a strong yet reversible complex. This complex formation provided an effective means of active tumor targeting of TAT-Gel, by 1) directing the TAT-Gel to CEA overexpressed tumor cells and 2) preventing nonspecific cell transduction to non-targeted normal cells. The cell transduction of TAT-Gel could, however, be efficiently reversed by addition of protamine. Feasibility of in vivo tumor targeting and "protamine-induced release" of TAT-Gel from the T84.66-Hep counterpart was confirmed by biodistribution and preliminary efficacy studies. CONCLUSIONS: This study successfully demonstrated in vitro and in vivo the applicability of PTD-modified ATTEMPTS for toxin-based cancer therapy.
Assuntos
Antineoplásicos/uso terapêutico , Pró-Fármacos/metabolismo , Animais , Antineoplásicos/farmacologia , Antígeno Carcinoembrionário/metabolismo , Linhagem Celular Tumoral , Sistemas de Liberação de Medicamentos , Heparina/administração & dosagem , Heparina/uso terapêutico , Humanos , Camundongos , Camundongos Nus , Proteínas Mutantes Quiméricas , Protaminas/administração & dosagem , Protaminas/uso terapêutico , Proteínas/metabolismo , Proteínas Inativadoras de Ribossomos Tipo 1/farmacologia , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Distribuição Tecidual , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
PURPOSE: A novel PEGylated and heparinized magnetic iron oxide nano-platform (DNPH) was synthesized for simultaneous magnetic resonance imaging (MRI) and tumor targeting. METHODS: Starch-coated magnetic iron oxide nanoparticles ("D") were crosslinked, aminated (DN) and then simultaneously PEGylated and heparinized with different feed ratios of PEG and heparin (DNPH1-4). DNPH products were characterized by Fourier transform infrared spectroscopy (FTIR), transmission electron microscopy (TEM) and superconducting quantum interference device (SQUID). The magentic targeting of DNPH3, with appropriate amounts of conjugated PEG and heparin, in a mouse 9L-glioma subcutaneous tumor model was confirmed by magnetic resonance imaging (MRI)/electron spin resonance (ESR). RESULTS: DNPH3 showed long circulating properties in vivo (half-life >8 h, more than 60-fold longer than that of parent D) and low reticuloendothelial system (RES) recognition in liver and spleen. Protamine, a model cationic protein, was efficiently loaded onto DNPH3 with a maximum loading content of 26.4 µg/mg Fe. Magnetic capture of DNPH3 in tumor site with optimized conditions (I.D. of 12 mg/kg, targeting time of 45 min) was up to 29.42 µg Fe/g tissue (12.26% I.D./g tissue). CONCLUSION: DNPH3 showed the potential to be used as a platform for cationic proteins for simultaneous tumor targeting and imaging.
Assuntos
Compostos Férricos/química , Glioma/diagnóstico , Heparina , Nanopartículas , Animais , Sistemas de Liberação de Medicamentos , Compostos Férricos/farmacocinética , Heparina/química , Heparina/farmacocinética , Imageamento por Ressonância Magnética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Nanopartículas/química , Nanopartículas/metabolismo , Polietilenoglicóis/química , Polietilenoglicóis/farmacocinética , Protaminas/administração & dosagemRESUMO
Starch-coated, PEGylated, and heparin-functionalized iron oxide magnetic nanoparticles (DNPH) were successfully synthesized and characterized in detail. The PEGylation (20 kDa) process resulted in an average coating of 430 PEG molecules per nanoparticle. After that, heparin conjugation was carried out to attain the final DNPH platform with 35.4 µg of heparin/mg of Fe. Commercially acquired heparin-coated magnetic nanoparticles were also PEGylated (HP) and characterized for comparison. Protamine was selected as a model protein to demonstrate the strong binding affinity and high loading content of DNPH for therapeutically relevant cationic proteins. DNPH showed a maximum loading of 22.9 µg of protamine/mg of Fe. In the pharmacokinetic study, DNPH displayed a long-circulating half-life of 9.37 h, 37.5-fold longer than that (0.15 h) of HP. This improved plasma stability enabled extended exposure of DNPH to the tumor lesions, as was visually confirmed in a flank 9L-glioma mouse model using magnetic resonance imaging (MRI). Quantitative analysis of the Fe content in excised tumor lesions further demonstrated the superior tumor targeting ability of DNPH, with up to 31.36 µg of Fe/g of tissue (13.07% injected dose (I.D.)/g of tissue) and 7.5-fold improvement over that (4.27 µg of Fe/g of tissue; 1.78% I.D./g of tissue) of HP. Overall, this study shed light on the potential of DNPH to be used as a protein drug delivery platform.
Assuntos
Sistemas de Liberação de Medicamentos/métodos , Heparina/química , Imageamento por Ressonância Magnética/métodos , Nanopartículas/química , Animais , Compostos Férricos/química , Glioma/diagnóstico , Glioma/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Nanopartículas/metabolismo , Protaminas/químicaRESUMO
In this review, we discussed the establishment of a so-called "theranostic" system by instituting the basic principles including the use of: [1] magnetic iron oxide nanoparticles (MION)-based drug carrier; [2] intra-arterial (I.A.) magnetic targeting; [3] macromolecular drugs with unmatched therapeutic potency and a repetitive reaction mechanism; [4] cell-penetrating peptide-mediated cellular drug uptake; and [5] heparin/protamine-regulated prodrug protection and tumor-specific drug re-activation into one single drug delivery system to overcome all possible obstacles, thereby achieving a potentially non-invasive, magnetic resonance imaging-guided, clinically enabled yet minimally toxic brain tumor drug therapy. By applying a topography-optimized I.A. magnetic targeting to dodge rapid organ clearance of the carrier during its first passage into the circulation, tumor capture of MION was enriched by >350 folds over that by conventional passive enhanced permeability and retention targeting. By adopting the prodrug strategy, we observed by far the first experimental success in a rat model of delivering micro-gram quantity of the large ß-galactosidase model protein selectively into a brain tumor but not to the ipsi- or contra-lateral normal brain regions. With the therapeutic regimens of most toxin/siRNA drugs to fully (>99.9%) eradicate a tumor being in the nano-molar range, the prospects of reaching this threshold become practically accomplishable.
Assuntos
Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/patologia , Sistemas de Liberação de Medicamentos/métodos , Imageamento por Ressonância Magnética/métodos , Nanopartículas de Magnetita , Animais , Portadores de Fármacos/administração & dosagem , Portadores de Fármacos/química , Portadores de Fármacos/farmacocinética , Humanos , Injeções Intra-Arteriais , Injeções Intravenosas , Nanopartículas de Magnetita/administração & dosagem , Nanopartículas de Magnetita/uso terapêuticoRESUMO
Directed enzyme/prodrug therapy (DEPT) has promising application for cancer therapy. However, most current DEPT strategies face shortcomings such as the loss of enzyme activity during preparation, low delivery and transduction efficiency in vivo and difficultly of monitoring. In this study, a novel magnetic directed enzyme/prodrug therapy (MDEPT) was set up by conjugating ß-glucosidase (ß-Glu) to aminated, starch-coated, iron oxide magnetic iron oxide nanoparticles (MNPs), abbreviated as ß-Glu-MNP, using glutaraldehyde as the crosslinker. This ß-Glu-MNP was then characterized in detail by size distribution, zeta potential, FTIR spectra, TEM, SQUID and magnetophoretic mobility analysis. Compared to free enzyme, the conjugated ß-Glu on MNPs retained 85.54% ± 6.9% relative activity and showed much better temperature stability. The animal study results showed that ß-Glu-MNP displays preferable pharmacokinetics characteristics in relation to MNPs. With an adscititious magnetic field on the surface of a tumor, a significant quantity of ß-Glu-MNP was selectively delivered into a subcutaneous tumor of a glioma-bearing mouse. Remarkably, the enzyme activity of the delivered ß-Glu in tumor lesions showed as high as 20.123±5.022 mU g(-1) tissue with 2.14 of tumor/non-tumor ß-Glu activity.
Assuntos
Enzimas Imobilizadas/metabolismo , Compostos Férricos/química , Magnetismo/métodos , Nanopartículas/química , Neoplasias/metabolismo , beta-Glucosidase/metabolismo , Animais , Espectroscopia de Ressonância de Spin Eletrônica , Fenômenos Magnéticos , Imageamento por Ressonância Magnética , Camundongos , Camundongos Endogâmicos C57BL , Nanopartículas/ultraestrutura , Neoplasias/patologia , Tamanho da Partícula , Espectroscopia de Infravermelho com Transformada de Fourier , Eletricidade Estática , beta-Glucosidase/farmacocinéticaRESUMO
Cocaine esterase (CocE) is the most efficient cocaine-metabolizing enzyme tested in vivo to date, displaying a rapid clearance of cocaine and a robust protection against cocaine's toxicity. Two potential obstacles to the clinical application of CocE, however, lie in its proteolytic degradation and induced immune response. To minimize these potential obstacles, we attempted nondisruptive cell encapsulation by creating a cell permeable form of CocE, which was achieved by covalently linking a thermally stable CocE mutant (dmCocE) with cell penetrating peptides (CPPs). Two types of CPPs, Tat and the low molecular weight protamine (LMWP), were used in this study. Two types of disulfide-bridged chemical conjugates, Tat-S-S-dmCocE and LMWP-S-S-dmCocE, were synthesized and then purified by heparin affinity chromatography. In addition, four recombinant CPP-dmCocE fusion proteins, Tat-N-dmCocE, LMWP-N-dmCocE, dmCocE-C-Tat, and dmCocE-C-LMWP, were constructed, expressed in Escherichia coli, and purified as soluble proteins. Among these six CPP-dmCocE variants, LMWP-S-S-dmCocE showed the highest cocaine-hydrolyzing activity, and dmCocE-C-Tat had the highest production yield. To evaluate their cellular uptake behavior, a covalently linked fluorophore (FITC) was utilized to visualize the cellular uptake of all six CPP-dmCocE variants in living HeLa cells. All the six variants exhibited cellular uptake, but their intracellular distribution phenotypes differed. While the chemical conjugates showed primarily cytoplasmic distribution, which was likely due to the reduction of the disulfide linkage between CPP and dmCocE, all the other four recombinant fusion proteins displayed both nuclear and cytoplasmic localization, with dmCocE-C-CPP exhibiting higher cytoplasmic distribution during cellular uptake. Based on a balanced consideration of essentials for clinical application, including parameters such as high cocaine-hydrolyzing efficiency, large production yield, major cytoplasmic distribution, etc., the dmCocE-C-Tat fusion protein seems to be the best candidate from this investigation. Further in vivo studies of the cell-encapsulated dmCocE-C-Tat in hydrolyzing cocaine and alleviating immunogenicity and proteolytic degradation in established, clinically relevant mouse models are currently underway in our laboratories. Findings from this research are not only useful for developing other new CPP-CocE constructs but also valuable for establishing a nondisruptive cell-encapsulation technology for other protein therapeutics that are known to be immunogenic for direct clinical application.
Assuntos
Hidrolases de Éster Carboxílico/química , Hidrolases de Éster Carboxílico/metabolismo , Peptídeos Penetradores de Células/química , Hidrolases de Éster Carboxílico/genética , Cromatografia de Afinidade , Células HeLa , Humanos , Fragmentos de Peptídeos/química , Protaminas/química , Recombinação Genética/genéticaRESUMO
BACKGROUND: Statin is a specific inhibitor of 3-hydroxy-3-methyl-glutaryl coenzyme A (HMG-CoA) reductase, a rate-limiting enzyme involved in the cholesterol synthesis pathway. In addition to their long-known efficacy for lowering cholesterol, statins have also been reported to possess anabolic effects on bone. Simvastatin is reported to increase cancellous bone volume, bone formation rate, and cancellous bone compressive strength in vivo. MATERIALS AND METHODS: In this report, the effects of simvastatin on osteoprecursor cells were evaluated. The effect on cell viability was determined by MTT assay, whereas differentiation and mineralization were examined using an alkaline phosphatase activity (ALP) test and alizarin red-S staining. Protein expressions related to bone formation, such as estrogen receptor-alpha (ER-α) and beta (ER-ß), were evaluated by using a Western blot analysis. To assess whether the osteoinductive effect of simvastatin occurs via estrogen receptor pathway, estrogen receptor agonist (E2) and antagonists (ICI 182,780) were applied to the cultures. RESULTS: Cultures grown in the presence of simvastatin exhibited an increased value for ALP activity and mineralization. The results of the Western blot analysis indicated that the addition of simvastatin up-regulated ER-α and ER-ß expression with a statistically significant difference in ER-α expression. Treatment of E2 led to an increase of the ALP activity and mineralization, but addition of the estrogen receptor antagonist ICI 182,780 revealed a decrease in both values. CONCLUSIONS: Based on these findings, it was concluded that simvastatin could produce positive effects on both the differentiation and mineralization of osteoprecursor cells. Our results also suggested that osteoinductive effects of simvastatin were achieved through ER pathway via the increase of ER-α expression.
Assuntos
Anticolesterolemiantes/farmacologia , Calcificação Fisiológica/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Receptor alfa de Estrogênio/metabolismo , Osteoblastos/efeitos dos fármacos , Sinvastatina/farmacologia , Fosfatase Alcalina/metabolismo , Animais , Western Blotting , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Receptor beta de Estrogênio/metabolismo , Camundongos , Osteoblastos/metabolismoRESUMO
Introduction: In targeted enzyme prodrug constructs, it is critical to control the bioactivity of the drug in its prodrug form. The preparation of such constructs often involves conjugation reactions directed to functional groups on amino acid side chains of the protein, which result in random conjugation and incomplete control of bioactivity of a prodrug, which may result in significant nontarget effect. Thus, more specific method of modification is desired. If the drug is a glycoprotein, enzymatic oxidation may offer an alternative approach for therapeutic glycoproteins. Methods: Tissue plasminogen activator (tPA), a model glycoprotein enzyme, was treated with galactose oxidase (GO) and horseradish peroxidase, followed by thiolation reaction and conjugation with low molecular weight heparin (LMWH). The LMWH-tPA conjugate was isolated by ion-exchange chromatography followed by centrifugal filtration. The conjugate was characterized for its fibrinolytic activity and for its plasminogen activation through an indirect amidolytic assay with a plasmin-specific substrate S-2251 when LMWH-tPA conjugate is complexed with protamine-albumin conjugate, followed by triggered activation in the presence of heparin. Results: LMWH-tPA conjugate prepared via enzymatic oxidation retained ~95% of its fibrinolytic activity with respect to native tPA. Upon complexation with protamine-albumin conjugate, the activity of LMWH-tPA was effectively inhibited (~90%) whereas the LMWH-tPA prepared by random thiolation exhibited ~55% inhibition. Addition of heparin fully generated the activities of both conjugates. Conclusion: The tPA was successfully modified via enzymatic oxidation by GO, resulting in enhanced control of its activity in the prodrug construct. This approach can be applied to other therapeutic glycoproteins.
RESUMO
In this study, a cell-penetrating peptide, the transactivating transcriptional factor (TAT) domain from HIV, was linked to a chitosan/doxorubicin (chitosan/DOX) conjugate to form a chitosan/DOX/TAT hybrid. The synthesized chitosan/DOX/TAT conjugate showed a different intracellular distribution pattern from a conjugate without TAT. Unlike both free DOX and the conjugate without TAT, the chitosan/DOX/TAT conjugate was capable of efficient cell entry. The chitosan/DOX/TAT conjugate was found to be highly cytotoxic, with an IC(50) value of approximately 480 nM, 2 times less than that of chitosan/DOX (980 nM). The chitosan/DOX/TAT provided decreases in tumor volume of 77.4 and 57.5% compared to free DOX and chitosan/DOX, respectively, in tumor-bearing mice. Therefore, this study suggests that TAT-mediated chitosan/DOX conjugate delivery is effective in slowing tumor growth.
Assuntos
Quitosana/uso terapêutico , Doxorrubicina/uso terapêutico , Neoplasias Experimentais/tratamento farmacológico , Fatores de Transcrição/uso terapêutico , Animais , Quitosana/farmacocinética , Doxorrubicina/farmacocinética , Eletroforese em Gel de Poliacrilamida , Feminino , Citometria de Fluxo , Camundongos , Camundongos Endogâmicos BALB C , Microscopia ConfocalRESUMO
The tissue-specific targeted delivery and efficient cellular uptake of siRNAs are the main obstacles to their clinical application. Antibody-siRNA-conjugates (ARCs) can deliver siRNA by exploiting the targeting property of antibodies like antibody-drug conjugates (ADCs). However, the effective conjugation of antibodies and siRNAs and the release of siRNAs specifically at target sites have posed challenges to the development of ARCs. In this study, the successful conjugation of antibodies and siRNAs was achieved using a multifunctional peptide as a linker, composed of a cell-penetrating peptide (CPP) and a substrate peptide (SP), which is highly expressed in solid tumors. The resulting antibody-multifunctional peptide (SP-CPP)-siRNA system delivered the siRNA to target tumor cells by the specific binding of the antibody. Once the enzymes on the tumor cell surface hydrolyzed the substrate peptide linker, siRNA-CPP was released from ARCs. The released siRNA-CPP entered the targeted cells via the cellular penetrating ability of CPP, resulting in improved siRNA-mediated gene silencing efficiency, verified both in vitro and in vivo. After intravenous administration, the designed ARCs achieved approximately 66.7% EGFP (Enhanced Green Fluorescent Protein) downregulation efficiency in nude mice xenografted with the HCT116-EGFP tumor model. The proposed system provides a prospective choice for ARC production and the safe and efficient delivery of siRNAs.
Assuntos
Peptídeos Penetradores de Células , Imunoconjugados , Animais , Linhagem Celular Tumoral , Camundongos , Camundongos Nus , Estudos Prospectivos , RNA Interferente PequenoRESUMO
Magnetic nanoparticles (MNP) have been widely studied for use in targeted drug delivery. Analysis of MNP biodistribution is essential to evaluating the success of targeting strategies and the potential for off-target toxicity. This work compared the applicability of inductively coupled plasma optical emission spectroscopy (ICP-OES) and electron spin resonance (ESR) spectroscopy in assessing MNP biodistribution. Biodistribution was evaluated in 9L-glioma bearing rats administered with MNP (12-25 mg Fe/kg) under magnetic targeting. Ex vivo analysis of MNP in animal tissues was performed with both ICP-OES and ESR. A cryogenic method was developed to overcome the technical hurdle of loading tissue samples into ESR tubes. Comparison of results from the ICP-OES and ESR measurements revealed two distinct relationships for organs accumulating high or low levels of MNP. In organs with high MNP accumulation such as the liver and spleen, data were strongly correlated (r = 0.97, 0.94 for the liver and spleen, respectively), thus validating the equivalency of the two methods in this high concentration range (>1000 nmol Fe/g tissue). The two sets of measurements, however, differed significantly in organs with lower levels of MNP accumulation such as the brain, kidney, and the tumor. Whereas ESR resolved MNP to 10-55 nmol Fe/g tissue, ICP-OES failed to detect MNP because of masking by endogenous iron. These findings suggest that ESR coupled to cryogenic sample handling is more robust than ICP-OES, attaining better sensitivity in analyses. Such advantages render ESR the method of choice for accurate profiling of MNP biodistribution across tissues with high variability in nanoparticle accumulation.
Assuntos
Espectroscopia de Ressonância de Spin Eletrônica/métodos , Compostos Férricos/química , Nanopartículas/química , Espectrofotometria Atômica/métodos , Animais , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Espectroscopia de Ressonância Magnética , Masculino , Ratos , Ratos Endogâmicos F344RESUMO
T84.66 is a monoclonal antibody with high affinity and specificity for tumor-associated carcinoembryonic antigen (CEA). In this work, we have developed an enzyme linked immunosorbent assay to determine T84.66 concentrations in mouse plasma. The assay was validated with respect to precision and accuracy by evaluating the recovery of T84.66 from mouse plasma. The working range of the assay is 25-200 ng/mL, and the limit of quantification is 2.5 microg/mL. Intra-assay recoveries ranged from 90.6 to 97.4%, and intra-assay precision reported as the percent coefficient of variation (CV%), ranged from 4.58 to 12.6%. Inter-assay recoveries were between 92.6 to 98.1% and the CV% ranged from 4.9-6.5%. The assay was tested for possible interference from soluble CEA. Soluble CEA, at concentrations up to 5 ng/mL, did not influence the recovery of T84.66. The assay was applied to study the pharmacokinetics of T84.66 in athymic Fox(nu) mice.
Assuntos
Anticorpos Monoclonais/sangue , Antígeno Carcinoembrionário/imunologia , Ensaio de Imunoadsorção Enzimática/métodos , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacocinética , Limite de Detecção , Camundongos , Camundongos Nus , Reprodutibilidade dos TestesRESUMO
Critical limb ischemia (CLI) is the most advanced stage of peripheral artery disease, associated with significant risk of limb loss, morbidity and mortality; however, there remains unmet therapeutic needs for arterial revascularization and ischemic tissue repair. Stem cell therapies have emerged as compelling candidates due to beneficial proangiogenic and immunosuppressive function. Nevertheless, in vivo efficacy was insufficient in proliferation, differentiation and survival/engraftment rate. Cardiac stem cells (CSCs) was firstly attempted for CLI as a novel therapeutic modality to provide superior angiogenic potency to bone marrow-derived stem cells (BMSCs). It was noted that CSCs demonstrated 3.2-fold in HGF, 2.9-fold in VEGF and 8.7-fold in PDGF-B higher gene expressions compared to BMSCs. To enhance the hypoxia-induced proangiogenic effect, CSCs were transfected with hypoxia-inducible factor-1 alpha (HIF-1α) by using electroporation method, specifically optimized for CSCs yielding 45.77% of transfection efficiency and 89.75% of viability. HIF-1α overexpression significantly increased CSC survival in hypoxia, proangiogenic factors production and endothelial differentiation. In mouse hind limb ischemia model, local intramuscular delivery of CSC overexpressing HIF-1α (HIF-CSC) significantly improved the blood flow recovery. Histological analysis revealed that muscle degeneration and fibrosis in the ischemic limb were attenuated. Local delivery of HIF-CSC might be a promising option for ischemic tissue restoration.
Assuntos
Células-Tronco Mesenquimais , Doença Arterial Periférica , Animais , Diferenciação Celular , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Isquemia/terapia , Camundongos , Neovascularização Patológica , Neovascularização Fisiológica , Doença Arterial Periférica/terapiaRESUMO
Overlapping substrate specificities within the family of matrix metalloproteinases (MMPs), usually caused by their highly conserved structural topology, increase the potential for a substrate to be cleaved by multiple enzymes within this family, which leads to the decrease in the selectivity of MMP substrate-based probes. To resolve this issue, MT1-MMP activatable fluorogenic probes for tumor detection with enhanced specificity were developed by combining a fluorescence resonance energy transfer (FRET) peptide substrate and its specific binding peptide with different lengths of linkers. The specificity of the probes increased profiting from the high affinity of the MT1-MMP specific binding peptide while keeping the ability to amplify the output imaging signals in response to MMP activity with the FRET substrate. Enzyme kinetics analysis clearly demonstrated that the conjugation of P-1 and MT1-AF7p enhanced both the specificity and selectivity of the fluorogenic probes for MT1-MMP, and introducing a linker composed of 12 PEG subunits into these two fragments led to optimized specificity and selectivity of the fluorogenic probe for MT1-MMP. Both in vitro and in vivo results revealed that the imaging probe with the linker composed of 12 PEG subunits based on our designed strategy could be effectively applied for MT1-MMP positive tumor imaging. Since this strategy for enhancing the specificity of protease sensing probes can be applied to other proteases and is not just limited to MT1-MMP, it is an appealing platform to achieve selective tumor imaging.
Assuntos
Corantes Fluorescentes/administração & dosagem , Metaloproteinase 14 da Matriz/administração & dosagem , Peptídeos/administração & dosagem , Animais , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Transferência Ressonante de Energia de Fluorescência , Corantes Fluorescentes/química , Humanos , Metaloproteinase 14 da Matriz/genética , Camundongos Endogâmicos BALB C , Camundongos Nus , Neoplasias/diagnóstico por imagem , Neoplasias/metabolismo , Peptídeos/química , Proteínas Recombinantes/administração & dosagemRESUMO
Non-injectable delivery of peptides and proteins is not feasible due to the limitations of large molecular mass, high hydrophilic properties, and gastrointestinal degradation. Therefore, proposing a new method to solve this problem is a burning issue. The objective of this study was to propose a novel protein delivery strategy to overcome the poor efficacy and irritation of buccal insulin delivery. In this study, we applied a conjugate of cell-penetrating peptides (LMWP) and insulin (INS-PEG-LMWP) for buccal delivery. INS-PEG-LMWP was prepared using insulin solution and mixture as references. The transport behaviour, in vivo bioactivity, hypoglycaemic effect, and safety of INS-PEG-LMWP were systematically characterised. An in vitro study demonstrated that the uptake and transportation of INS-PEG-LMWP across buccal mucosal multilayers significantly increased. By comparing the effects of different endocytic inhibitors on INS-PEG-LMWP uptake, the conjugate might be delivered via an energy independent, electrostatically adsorbed pathway. INS-PEG-LMWP's relative pharmacological bioavailability was high and its relative bioavailability was up to 26.86%, demonstrating no visible mucosal irritation. Cell-penetrating peptides are likely to become a reliable and safe tool for overcoming insulin's low permeability through the epithelial multilayers, the major barrier to buccal delivery.
Assuntos
Peptídeos Penetradores de Células/administração & dosagem , Hipoglicemiantes/administração & dosagem , Insulina/administração & dosagem , Mucosa Bucal/metabolismo , Polietilenoglicóis/administração & dosagem , Animais , Linhagem Celular Tumoral , Peptídeos Penetradores de Células/farmacocinética , Humanos , Hipoglicemiantes/sangue , Hipoglicemiantes/farmacocinética , Insulina/sangue , Insulina/farmacocinética , Masculino , Absorção pela Mucosa Oral , Permeabilidade , Polietilenoglicóis/farmacocinética , Coelhos , SuínosRESUMO
Carcinoembryonic antigen (CEA) is a tumor associated antigen that is over-expressed in colorectal cancer and several other cancers of the gastrointestinal system. An enzyme linked immunosorbent assay was developed to determine CEA concentrations in mouse plasma. The assay was validated over the standard curve range of 1-20 ng/mL. The intra-assay recoveries ranged from 93-104% with associated percent coefficients of variation (CV%) ranging between 2.5-12.8%. The inter-assay recoveries were in the range of 98.4-105% and their CV% values were between 4.77-10.1%. The assay was used to detect the presence of circulating CEA in the LS174T adenocarcinoma xenograft model and to study the pharmacokinetics of recombinant CEA in athymic mice.
Assuntos
Adenocarcinoma/sangue , Antígeno Carcinoembrionário/sangue , Neoplasias Colorretais/sangue , Ensaio de Imunoadsorção Enzimática , Animais , Linhagem Celular Tumoral , Humanos , Masculino , Camundongos , Camundongos Nus , Transplante de Neoplasias , Sensibilidade e EspecificidadeRESUMO
Integrating the functions of bioimaging, targeting and controlled release of therapeutic agents into a single nanoparticle is of great interests in nanomedicine and nanobiology. Herein, a cis -diol/pH dual-responsive upconversion nanoparticle (UCNP)-based theranostic platform has been developed for delivery of the anticancer drug to cancer cells. This nanoplatform is based on the strategic design of targetable hyaluronan modified UCNPs (HA-UCNPs) that are coupled with aminobenzeneboronic acid (APBA) to obtain APBA-UCNPs, having favorable tumor selectivity as well as the capacity for capturing cis-diol-containing therapeutics. The controlled release function is then achieved through the self-assembly of hydroxycamptothecin derivative ligands onto the surfaces of APBA-UCNPs, which is controllable in a stimuli-dependent manner. The UCNP-based theranostic probe taken up by tumor cells via receptor-mediated endocytosis liberates drugs triggered by competitive glucose at low pH in endosomes/lysosomes, resulting in cell apoptosis. The dual-responsive mechanism of boronate ester bonds gives a chemoselective strategy for controlled release of drug within tumor cells, establishing an alternative approach to treat a broad spectrum of diseases exploiting similar boronic acid-involved therapeutics.