RESUMO
This study aimed to control the oral absorption of cyclosporine A (CsA) with the use of a mucosal drug delivery system (mDDS). Mucopenetrating nanocarriers (MP/NCs) and mucoadhesive nanocarriers (MA/NCs) were prepared by flash nanoprecipitation employing polystyrene-block-poly(ethylene glycol) and polystyrene-block-poly(N,N-dimethyl aminoethyl methacrylate), respectively. Their particle distribution in the rat gastrointestinal tract were visualized by fluorescent imaging. Plasma concentrations were monitored after oral administration of CsA-loaded MP/NCs (MP/CsA) and MA/NCs (MA/CsA) to rats. MP/NCs and MA/NCs had a particle size below 200 nm and ζ-potentials of 4 and 40 mV, respectively. The results from in vitro experiments demonstrated mucopenetration of MP/NCs and mucoadhesion of MA/NCs. Confocal laser scanning microscopic images showed diffusion of MP/NCs in the gastrointestinal mucus towards epithelial cells and localization of MA/NCs on the surface of the gastrointestinal mucus layer. In a pH 6.8 solution, rapid and sustained release of CsA were observed for MP/CsA and MA/CsA, respectively. After oral dosing (10 mg-CsA/kg) to rats, amorphous CsA powder exhibited a time to maximum plasma concentration (Tmax) of 3.4 h, maximum plasma concentration (Cmax) of 0.12 µg/mL, and bioavailability of 0.7%. Compared with amorphous CsA powder, MP/CsA shortened Tmax by 1.1 to 2.3 h and increased the bioavailability by 43-fold to 30.1%, while MA/CsA prolonged Tmax by 3.4 to 6.8 h with Cmax and bioavailability of 0.65 µg/mL and 11.7%, respectively. These pharmacokinetic behaviors would be explained by their diffusion and release properties modulated by polymeric surface modification. The mDDS approach is a promising strategy for the pharmacokinetic control of orally administered CsA.
Assuntos
Ciclosporina , Sistemas de Liberação de Medicamentos , Ratos Sprague-Dawley , Animais , Ciclosporina/farmacocinética , Ciclosporina/administração & dosagem , Masculino , Administração Oral , Ratos , Imunossupressores/farmacocinética , Imunossupressores/administração & dosagem , Imunossupressores/sangue , Portadores de Fármacos/química , Mucosa Intestinal/metabolismo , Nanopartículas/química , Disponibilidade Biológica , Tamanho da PartículaRESUMO
PURPOSE: This study was undertaken to develop novel mucoadhesive formulations of clofazimine (CFZ), a drug candidate for the treatment of cryptosporidiosis, with the aim of strategic delivery to the small intestine, the main site of the disease parasites. METHODS: CFZ-loaded nanoparticles (nCFZ) coated with non-biodegradable anionic polymer (nCFZ/A) and biodegradable anionic protein complex (nCFZ/dA) were prepared by Flash NanoPrecipitation (FNP) and evaluated for their physicochemical and biopharmaceutical properties. RESULTS: The mean diameters of nCFZ/A and nCFZ/dA were ca. 90 and 240 nm, respectively, and they showed narrow size distributions and negative ζ-potentials. Both formulations showed higher solubility of CFZ in aqueous solution than crystalline CFZ. Despite their improved dispersion behaviors, both formulations exhibited significantly lower diffusiveness than crystalline CFZ in a diffusion test using artificial mucus (AM). Quartz crystal microbalance analysis showed that both formulations clearly interacted with mucin, which appeared to be responsible for their reduced diffusiveness in AM. These results suggest the potent mucoadhesion of nCFZ/A and nCFZ/dA. After the oral administration of CFZ samples (10 mg-CFZ/kg) to rats, nCFZ/dA and nCFZ/A exhibited a prolongation in Tmax by 2 and >9 h, respectively, compared with crystalline CFZ. At 24 h after oral doses of nCFZ/A and nCFZ/dA with mucoadhesion, there were marked increases in the intestinal CFZ concentration (4-7 fold) compared with Lamprene®, a commercial CFZ product, indicating enhanced CFZ exposure in the small intestine. CONCLUSION: The use of FNP may produce mucoadhesive CFZ formulations with improved intestinal exposure, possibly offering enhanced anti-cryptosporidium therapy.
Assuntos
Clofazimina/administração & dosagem , Sistemas de Liberação de Fármacos por Nanopartículas/química , Administração Oral , Animais , Clofazimina/farmacocinética , Criptosporidiose/tratamento farmacológico , Liberação Controlada de Fármacos , Humanos , Absorção Intestinal , Mucosa Intestinal/metabolismo , Intestino Delgado/metabolismo , Masculino , Modelos Animais , Ratos , SolubilidadeRESUMO
Because of its attractive cost and yield, hierarchical assembly, in which constituent structures of lower hierarchy share a majority of components, is an appealing approach to scale up DNA self-assembly. A few strategies have already been investigated to combine preformed DNA nanostructures. In this study, we present a new hierarchical assembly method based on four-way toehold-mediated strand displacement to facilitate the combination of preformed DNA structural units. Employing such a method, we have constructed a series of higher-order structures composed of 5, 7, 9, 11, 13, and 15 preformed units respectively.
RESUMO
Nanoprecipitation of active pharmaceutical ingredients (APIs) to form nanocarriers (NCs) is an attractive method of producing formulations with improved stability and biological efficacies. However, nanoprecipitation techniques have not been demonstrated for highly soluble peptide therapeutics. We here present a model and technique to encapsulate highly water-soluble biologic APIs by manipulating API salt forms. APIs are ion paired with hydrophobic counterions to produce new API salts that exhibit altered solubilities suitable for nanoprecipitation processing. The governing rules of ion pair identity and processing conditions required for successful encapsulation are experimentally determined and assessed with theoretical models. Successful NC formation for the antibiotic polymyxin B requires hydrophobicity of the ion pair acid to be greater than logP = 2 for strong acids and greater than logP = 8 for weak acids. Oleic acid with a logP = 8, and pKa = 5, appears to be a prime candidate as an ion pair agent since it is biocompatible and forms excellent ion pair complexes. NC formation from preformed, organic soluble ion pairs is compared to in situ ion pairs where NCs are made in a single precipitation step. NC properties, such as stability and release rates, can be tuned by varying ion pair molecular structure and ion pair-to-API molar ratios. For polymyxin B, NCs ≈ 100-200 nm in size, displaying API release rates over 3 days, were produced. This work demonstrates a new approach that enables the formation of nanoparticles from previously intractable compounds.
Assuntos
Antibacterianos/química , Portadores de Fármacos/química , Nanopartículas/química , Peptídeos/química , Preparações de Ação Retardada , Liberação Controlada de Fármacos , Interações Hidrofóbicas e Hidrofílicas , Polimixina B/química , SolubilidadeRESUMO
Clofazimine, a lipophilic (log P = 7.66) riminophenazine antibiotic approved by the US Food and Drug Administration (FDA) with a good safety record, was recently identified as a lead hit for cryptosporidiosis through a high-throughput phenotypic screen. Cryptosporidiosis requires fast-acting treatment as it leads to severe symptoms which, if untreated, result in morbidity for infants and small children. Consequently, a fast-releasing oral formulation of clofazimine in a water-dispersible form for pediatric administration is highly desirable. In this work, clofazimine nanoparticles were prepared with three surface stabilizers, hypromellose acetate succinate (HPMCAS), lecithin, and zein, using the flash nanoprecipitation (FNP) process. Drug encapsulation efficiencies of over 92% were achieved. Lyophilization and spray-drying were applied and optimized to produce redispersible nanoparticle powders. The release kinetics of these clofazimine nanoparticle powders in biorelevant media were measured and compared with those of crystalline clofazimine and the currently marketed formulation Lamprene. Remarkably improved dissolution rates and clofazimine supersaturation levels up to 90 times equilibrium solubility were observed with all clofazimine nanoparticles tested. Differential scanning calorimetry indicated a reduction of crystallinity of clofazimine in nanoparticles. These results strongly suggest that the new clofazimine nanoparticles prepared with affordable materials in this low-cost nanoparticle formulation process can be used as viable cryptosporidiosis therapeutics.
Assuntos
Antiparasitários/farmacologia , Clofazimina/farmacologia , Criptosporidiose/tratamento farmacológico , Portadores de Fármacos/química , Composição de Medicamentos/métodos , Desenho de Fármacos , Antiparasitários/economia , Antiparasitários/uso terapêutico , Varredura Diferencial de Calorimetria , Química Farmacêutica , Clofazimina/economia , Clofazimina/uso terapêutico , Cristalização , Dessecação , Portadores de Fármacos/economia , Composição de Medicamentos/economia , Liberação Controlada de Fármacos , Excipientes/química , Liofilização , Nanopartículas/química , Nanopartículas/economia , Tamanho da Partícula , Solubilidade , Fatores de TempoRESUMO
One factor limiting the expansion of nanomedicines has been the high cost of the materials and processes required for their production. We present a continuous, scalable, low cost nanoencapsulation process, Flash Nanoprecipitation (FNP) that enables the production of nanocarriers (NCs) with a narrow size distribution using zein corn proteins. Zein is a low cost, GRAS protein (having the FDA status of "Generally Regarded as Safe") currently used in food applications, which acts as an effective encapsulant for hydrophobic compounds using FNP. The four-stream FNP configuration allows the encapsulation of very hydrophobic compounds in a way that is not possible with previous precipitation processes. We present the encapsulation of several model active compounds with as high as 45 wt % drug loading with respect to zein concentration into â¼100 nm nanocarriers. Three examples are presented: (1) the pro-drug antioxidant, vitamin E-acetate, (2) an anticholera quorum-sensing modulator CAI-1 ((S)-3-hydroxytridecan-4-one; CAI-1 that reduces Vibrio cholerae virulence by modulating cellular communication), and (3) hydrophobic fluorescent dyes with a range of hydrophobicities. The specific interaction between zein and the milk protein, sodium caseinate, provides stabilization of the NCs in PBS, LB medium, and in pH 2 solutions. The stability and size changes in the three media provide information on the mechanism of assembly of the zein/active/casein NC.
Assuntos
Antioxidantes/química , Portadores de Fármacos/química , Nanopartículas/química , Zeína/química , Antioxidantes/farmacologia , Caseínas/química , Portadores de Fármacos/farmacologia , Interações Hidrofóbicas e Hidrofílicas , Nanomedicina , Nanopartículas/administração & dosagem , Tamanho da Partícula , Pró-Fármacos/química , Pró-Fármacos/farmacologia , Percepção de Quorum/efeitos dos fármacos , Vitamina E/química , Zea mays/química , Zeína/farmacologiaRESUMO
The rise of bacterial antibiotic resistance has created a demand for alternatives to traditional antibiotics. Attractive possibilities include pro- and anti-quorum sensing therapies that function by modulating bacterial chemical communication circuits. We report the use of Flash NanoPrecipitation to deliver the Vibrio cholerae quorum-sensing signal CAI-1 ((S)-3-hydroxytridecan-4-one) in a water dispersible form as nanoparticles. The particles activate V. cholerae quorum-sensing responses 5 orders of magnitude higher than does the identically administered free CAI-1 and are diffusive across in vivo delivery barriers such as intestinal mucus. This work highlights the promise of combining quorum-sensing strategies with drug delivery approaches for the development of next-generation medicines.
Assuntos
Biofilmes/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , Cetonas/administração & dosagem , Nanocápsulas/química , Percepção de Quorum/efeitos dos fármacos , Percepção de Quorum/fisiologia , Cetonas/química , Nanocápsulas/ultraestrutura , Tamanho da PartículaRESUMO
We have created a set of rationally designed peptides that form calcium-dependent hydrogels based on the beta roll peptide domain. In the absence of calcium, the beta roll domain is intrinsically disordered. Upon the addition of calcium, the peptide forms a beta helix secondary structure. We have designed two variations of our beta roll domain. First, we have mutated one face of the beta roll domain to contain leucine residues so that the calcium-dependent structural formation leads to dimerization through hydrophobic interactions. Second, an α-helical leucine zipper domain is appended to the engineered beta roll domain as an additional means of forming intermolecular cross-links. This full peptide construct forms a hydrogel only in calcium-rich environments. The resulting structural and mechanical properties of the supramolecular assemblies are compared with the wild-type domain using several biophysical techniques including circular dichroism, FRET, bis-ANS binding and microrheology. The calcium responsiveness and rheological properties of the leucine beta roll containing construct confirm the potential of this allosterically regulated scaffold to serve as a cross-linking domain for stimulus-responsive biomaterials development.
Assuntos
Cálcio/química , Reagentes de Ligações Cruzadas/síntese química , Hidrogel de Polietilenoglicol-Dimetacrilato/síntese química , Leucina/química , Peptídeos/síntese química , Engenharia de Proteínas , Reagentes de Ligações Cruzadas/química , Hidrogel de Polietilenoglicol-Dimetacrilato/química , Substâncias Macromoleculares/síntese química , Substâncias Macromoleculares/química , Modelos Moleculares , Peptídeos/química , Estrutura Secundária de ProteínaRESUMO
PURPOSE: Blood-brain barrier disruption (BBBD) is of interest for treating neurodegenerative diseases and tumors by enhancing drug delivery. Focused ultrasound (FUS) is a powerful method to alleviate BBB challenges; however, the detection of BBB opening by non-invasive methods remains limited. The purpose of this work is to demonstrate that 3D transcranial color Doppler (3DCD) and photoacoustic imaging (PAI) combined with custom-made nanoparticle (NP)-mediated FUS delivery can detect BBBD in mice. PROCEDURES: We use MRI and stereotactic ultrasound-mediated BBBD to create and confirm four openings in the left hemisphere and inject intravenously indocyanine green (ICG) and three sizes (40 nm, 100 nm, and 240 nm in diameter) of fluorophore-labeled NPs. We use PAI and fluorescent imaging (FI) to assess the spatial distribution of ICG/NPs in tissues. RESULTS: A reversible 41 ± 12 % (n = 8) decrease in diameter of the left posterior cerebral artery (PCA) relative to the right after FUS treatment is found using CD images. The spectral unmixing of photoacoustic images of the in vivo (2 h post FUS), perfused, and ex vivo brain reveals a consistent distribution pattern of ICG and NPs at *FUS locations. Ex vivo spectrally unmixed photoacoustic images show that the opening width is, on average, 1.18 ± 0.12 mm and spread laterally 0.49 ± 0.05 mm which correlated well with the BBB opening locations on MR images. In vivo PAI confirms a deposit of NPs in tissues for hours and potentially days, is less sensitive to NPs of lower absorbance at a depth greater than 3 mm and too noisy with NPs above an absorbance of 85.4. FI correlates well with ex vivo PAI to a depth of 3 mm in tissues for small NPs and 4.74 mm for large NPs. CONCLUSIONS: 3DCD can monitor BBBD over time by detecting reversible anatomical changes in the PCA. In vivo 3DPAI at 15 MHz combined with circulating ICG and/or NPs with suitable properties can assess BBB opening 2 h post FUS.
Assuntos
Barreira Hematoencefálica/efeitos dos fármacos , Artérias Cerebrais/diagnóstico por imagem , Sistemas de Liberação de Medicamentos , Nanopartículas/química , Técnicas Fotoacústicas , Ultrassonografia Doppler , Animais , Encéfalo/diagnóstico por imagem , Mapeamento Encefálico , Imageamento Tridimensional , Verde de Indocianina , Imageamento por Ressonância Magnética , Camundongos , Microbolhas , Microscopia de Fluorescência , Neoplasias/diagnóstico por imagem , Neoplasias/terapia , Doenças Neurodegenerativas/diagnóstico por imagem , Doenças Neurodegenerativas/terapiaRESUMO
The present study develops cyclosporine A (CsA)-loaded polymeric nanocarriers with mucus-diffusive and mucus-adhesive potential to control pharmacokinetic behavior after oral administration for the treatment of inflammatory bowel diseases (IBD). CsA-loaded nanocarriers consisting of polystyrene-block-polyethylene glycol (PEG-CsA) and polystyrene-block-polyacrylic acid (PAA-CsA) were prepared by a flash nanoprecipitation. Both nanocarriers showed redispersibility from lyophilized powder back to uniform nanocarrier with a mean diameter of approximately 150 nm. The nanocarriers exhibited significantly improved release behavior of CsA under pH 6.8 condition compared. A test of mucodiffusion, using artificial mucus, demonstrated the mucus-diffusive and mucus-adhesive potential of PEG-CsA and PAA-CsA, respectively, dependent on the lack of electrostatic interactions between the surface-coated polymer and mucin. Oral administrations of PEG-CsA and PAA-CsA (10 mg-CsA/kg) in rats resulted in significant improvements of absorption, as evidenced by 50- and 25-fold higher bioavailability than crude CsA, respectively. PAA-CsA exhibited more sustained and slower absorption process of CsA than PEG-CsA because of the different diffusion behavior within the mucus layer. In the rat model of IBD, significant suppression of inflammatory symptoms could be achieved by oral treatment with both CsA nanoparticles. These polymeric nanocarriers are promising dosage options to control pharmacokinetic behavior of orally dosed CsA, contributing to the development of safe and effective treatment for IBD.
Assuntos
Ciclosporina , Nanopartículas , Adesivos , Animais , Química Farmacêutica , Portadores de Fármacos , Muco , Tamanho da Partícula , Polímeros , RatosRESUMO
Short structured peptides can provide scaffolds for protease-resistant peptide therapeutics, serve as useful building blocks in biomedical and biotechnological applications, and shed light on the role of secondary structure elements in protein folding. It is well known that directed evolution is a powerful method for creating proteins and peptides with novel properties, and a system for the selection of short peptides based on structure from a randomized library would be an important advancement. In this study, phage particles monovalently displaying a short peptide and an N-terminal 6xHis tag on their P3 coat protein were bound to nickel agarose resin and were subsequently challenged with a protease that specifically cleaves at a site within the peptide. The extent to which phage is proteolytically released from the resin was found to be dependent on the structural properties of the inserted peptide sequences. As proofs-of-concept, a structured peptide has been isolated from a pool of flexible peptides using a trypsin selection, and a flexible peptide has been isolated from a pool of structured peptides using a chymotrypsin selection. This selection system will be a strong technological platform for the creation of short peptides with interesting structural properties using directed evolution.
Assuntos
Bacteriófagos/metabolismo , Peptídeos/metabolismo , Sequência de Aminoácidos , Quimotripsina/metabolismo , Biblioteca de Peptídeos , Peptídeos/química , Dobramento de Proteína , Estrutura Terciária de ProteínaRESUMO
While the formulation of nanoparticle (NP) suspensions has been widely applied in materials and life science, the recovery of NPs from such a suspension into a solid state is practically important to confer long-term storage stability. However, solidification, while preserving the original nanoscale properties, remains a formidable challenge in the pharmaceutical and biomedical applications of NPs. Herein we combined flash nanoprecipitation (FNP) and spray-drying as a nanofabrication platform for NP formulation and recovery without compromising the dissolution kinetics of the active ingredient. Clofazimine was chosen to be the representative drug, which has been recently repurposed as a potential treatment for cryptosporidiosis. Clofazimine was encapsulated in NPs with low-cost surface coatings, hypromellose acetate succinate (HPMCAS) and lecithin, which were required by the ultimate application to global health. Spray-drying and lyophilization were utilized to produce dried powders with good long-term storage stability for application in hot and humid climatic zones. The particle morphology, yield efficiency, drug loading, and clofazimine crystallinity in the spray-dried powders were characterized. The in vitro release kinetics of spray-dried NP powders were compared to analogous dissolution profiles from standard lyophilized NP samples, crystalline clofazimine powder, and the commercially available formulation Lamprene. The spray-dried powders showed a supersaturation level of up to 60 times the equilibrium solubility and remarkably improved dissolution rates. In addition, the spray-dried powders with both surface coatings showed excellent stability during aging studies with elevated temperature and humidity, in view of the dissolution and release in vitro. Considering oral delivery for pediatric administration, the spray-dried powders show less staining effects with simulated skin than crystalline clofazimine and may be made into minitablets without additional excipients. These results highlight the potential of combining FNP and spray-drying as a feasible and versatile platform to design and rapidly recover amorphous NPs in a solid dosage form, with the advantages of satisfactory long-term storage stability, low cost, and easy scalability.
RESUMO
Pseudomonas aeruginosa is an opportunistic gram-negative pathogen that causes a wide range of infections; it is becoming increasingly difficult to treat due to antibiotic resistance. Quorum-sensing (QS) based therapeutics, which function by disabling pathogen virulence without killing pathogens, are a promising class of drugs that may be used to treat bacterial infections without eliciting resistance development. The use of QS drugs to treat pulmonary P. aeruginosa infections, however, has been greatly limited due to the inability to deliver QS drugs at sufficiently high concentrations past physiological barriers such as pulmonary mucus. Here we apply a block copolymer-directed self-assembly process, Flash NanoPrecipitation, to develop a series of QS-active formulations that are fully water dispersible, stable, and mucus-penetrating. These formulations inhibit P. aeruginosa virulence without inhibiting cell growth. Particle size (70â¯nm-400â¯nm) and release rate (1â¯h-14â¯days) can be tuned by altering constructs' physical properties and formulation excipients. We also demonstrate, to the best of our knowledge, the first instance of a QS nanocarrier platform technology that can penetrate through human cystic fibrosis pulmonary mucus. This work highlights the need to incorporate nanoformulation strategies into the development of next-generation antimicrobial therapeutics.
Assuntos
Portadores de Fármacos/administração & dosagem , Nanopartículas/administração & dosagem , Polímeros/administração & dosagem , Pseudomonas aeruginosa/efeitos dos fármacos , Piocianina/metabolismo , Percepção de Quorum , Virulência/efeitos dos fármacos , Fibrose Cística/metabolismo , Portadores de Fármacos/química , Humanos , Muco/metabolismo , Nanopartículas/química , Polímeros/química , Pseudomonas aeruginosa/metabolismo , Pseudomonas aeruginosa/fisiologiaRESUMO
Malaria poses a major burden on human health and is becoming increasingly difficult to treat due to the development of antimalarial drug resistance. The resistance issue is further exacerbated by a lack of patient adherence to multi-day dosing regimens. This situation motivates the development of new antimalarial treatments that are less susceptible to the development of resistance. We have applied Flash NanoPrecipitation (FNP), a polymer-directed self-assembly process, to form stable, water-dispersible nanoparticles (NPs) of 50-400 nm in size containing OZ439, a poorly orally bioavailable but promising candidate for single-dose malaria treatment developed by Medicines for Malaria Venture (MMV). During the FNP process, a hydrophobic OZ439 oleate ion paired complex was formed and was encapsulated into NPs. Lyophilization conditions for the NP suspension were optimized to produce a dry powder. The in vitro release rates of OZ439 encapsulated in this powder were determined in biorelevant media and compared with the release rates of the unencapsulated drug. The OZ439 NPs exhibit a sustained release profile and several-fold higher release concentrations compared to that of the unencapsulated drug. In addition, XRD suggests the drug was stabilized into an amorphous form within the NPs, which may explain the improvement in dissolution kinetics. Formulating OZ439 into NPs in this way may be an important step toward developing a single-dose oral malaria therapeutic, and offers the possibility of reducing the amount of drug required per patient, lowering delivery costs, and improving dosing compliance.
Assuntos
Adamantano/análogos & derivados , Antimaláricos/administração & dosagem , Composição de Medicamentos , Nanopartículas/química , Peróxidos/administração & dosagem , Adamantano/administração & dosagem , Adamantano/química , Adamantano/farmacocinética , Antimaláricos/química , Antimaláricos/farmacocinética , Sistemas de Liberação de Medicamentos , Liberação Controlada de Fármacos , Humanos , Interações Hidrofóbicas e Hidrofílicas , Íons/química , Estrutura Molecular , Peróxidos/química , Peróxidos/farmacocinéticaRESUMO
Nanoparticles (NP) are promising contrast agents for positron emission tomography (PET) radionuclide imaging that can increase signal intensity by localizing clusters of PET radionuclides together. However, methods to load NPs with PET radionuclides suffer from harsh loading conditions or poor loading efficacies or result in NP surface modifications that alter targeting in vivo. We present the formation of water-dispersible, polyethylene glycol coated NPs that encapsulate phthalocyanines into NP cores at greater than 50 wt % loading, using the self-assembly technique Flash NanoPrecipitation. Particles from 70 to 160 nm are produced. Phthalocyanine NPs rapidly and spontaneously chelate metals under mild conditions and can act as sinks for PET radionuclides such as 64-Cu to produce PET-active NPs. NPs chelate copper(II) with characteristic rates of 1845 M-1 h-1 at pH 6 and 37 °C, which produced >90% radionuclide chelation within 1 h. NP physical properties, such as core composition, core fluidity, and size, can be tuned to modulate chelation kinetics. These NPs retain 64Cu even in the presence of the strong chelator ethylene diamine tetraacetic acid. The development of these constructs for rapid and facile radionuclide labeling expands the applications of NP-based PET imaging.
Assuntos
Nanopartículas , Cobre , Radioisótopos de Cobre , Tomografia por Emissão de PósitronsRESUMO
Optical imaging is a rapidly progressing medical technique that can benefit from the development of new and improved optical imaging agents suitable for use in vivo. However, the molecular rules detailing what optical agents can be processed and encapsulated into in vivo presentable forms are not known. We here present the screening of series of highly hydrophobic porphyrin, phthalocyanine, and naphthalocyanine dye macrocycles through a self-assembling Flash NanoPrecipitation process to form a series of water dispersible dye nanoparticles (NPs). Ten out of 19 tested dyes could be formed into poly(ethylene glycol) coated nanoparticles 60-150 nm in size, and these results shed insight on dye structural criteria that are required to permit dye assembly into NPs. Dye NPs display a diverse range of absorbance profiles with absorbance maxima within the NIR region, and have absorbance that can be tuned by varying dye choice or by doping bulking materials in the NP core. Particle properties such as dye core load and the compositions of co-core dopants were varied, and subsequent effects on photoacoustic and fluorescence signal intensities were measured. These results provide guidelines for designing NPs optimized for photoacoustic imaging and NPs optimized for fluorescence imaging. This work provides important details for dye NP engineering, and expands the optical imaging tools available for use.
Assuntos
Corantes Fluorescentes/química , Indóis/química , Nanopartículas/química , Porfirinas/química , Fluorescência , Isoindóis , Imagem Óptica/métodos , Tamanho da Partícula , Técnicas Fotoacústicas/métodosRESUMO
Photoacoustic (PA) imaging is a developing diagnostic technique where multiple species can be simultaneously imaged with high spatial resolution in 3D if the absorbance spectrum of each species is distinct and separable. However, multiplexed PA imaging has been greatly limited by the availability of spectrally separable contrast agents that can be used in vivo. Toward this end, we present the formation and application of a series of poly ethylene glycol (PEG)-coated nanoparticles (NPs) with unique separable absorbance profiles suitable for simultaneous multiplexed imaging. As a proof-of-concept, we demonstrate this form of mixed-sample multiplexed imaging, using cRGD peptide surface-modified NPs with nonmodified NPs in a murine subcutaneous Lewis lung carcinoma tumor model. The simultaneous imaging of nonmodified NPs provides an "internal standard", to deconvolute the contributions of active-ligand and passive-NP targeting effects. Particles with 25% surface cRGD modification display 52 ± 22 fold higher liver to tumor ratio accumulation levels, while the same set of particles display only 9.8 ± 4 fold accumulation levels when internally normalized. The pharmacokinetic profiles of targeted and nontargeted NPs can be simultaneously tracked in real-time to study how biodistribtions of particles are affected by ligand modification. The internal normalization of control particles greatly enhances the precision and decreases the number of animals needed in studies of nanoparticle targeting. These new dyes are an enabling technology for PA imaging of NP fate and targeting. This is the first demonstration of real-time multiplexed PA imaging of mixed-targeted samples in vivo.
RESUMO
Photoacoustic (PA) imaging is an emerging hybrid optical-ultrasound based imaging technique that can be used to visualize optical absorbers in deep tissue. Free organic dyes can be used as PA contrast agents to concurrently provide additional physiological and molecular information during imaging, but their use in vivo is generally limited by rapid renal clearance for soluble dyes and by the difficulty of delivery for hydrophobic dyes. We here report the use of the block copolymer directed self-assembly process, Flash NanoPrecipitation (FNP), to form series of highly hydrophobic optical dyes into stable, biocompatible, and water-dispersible nanoparticles (NPs) with sizes from 38 to 88 nm and with polyethylene glycol (PEG) surface coatings suitable for in vivo use. The incorporation of dyes with absorption profiles within the infrared range, that is optimal for PA imaging, produces the PA activity of the particles. The hydrophobicity of the dyes allows their sequestration in the NP cores, so that they do not interfere with targeting, and high loadings of >75 wt % dye are achieved. The optical extinction coefficients (ε (mL mg(-1) cm(-1))) were essentially invariant to the loading of the dye in NP core. Co-encapsulation of dye with vitamin E or polystyrene demonstrates the ability to simultaneously image and deliver a second agent. The PEG chains on the NP surface were functionalized with folate to demonstrate folate-dependent targeting. The spectral separation of different dyes among different sets of particles enables multiplexed imaging, such as the simultaneous imaging of two sets of particles within the same animal. We provide the first demonstration of this capability with PA imaging, by simultaneously imaging nontargeted and folate-targeted nanoparticles within the same animal. These results highlight Flash NanoPrecipitation as a platform to develop photoacoustic tools with new diagnostic capabilities.
Assuntos
Diagnóstico por Imagem/métodos , Nanopartículas/química , Técnicas Fotoacústicas , Animais , Raios Infravermelhos , Polietilenoglicóis/química , Polímeros/químicaRESUMO
Hydrogels are gaining attention as injectable vehicles for delivery of therapeutics for a range of applications. We describe self-assembling and injectable Dock-and-Lock hydrogels for local delivery of interleukin-10 (IL-10) to abate the progression of inflammation and fibrosis that leads to chronic kidney disease. As monitored with a fluorescent tag, hydrogels degraded within a few days in vitro and matched IL-10 release profiles; however, hydrogels remained in the kidney for up to 30 days in vivo. A unilateral ureteral obstruction (UUO) mouse model was used to investigate in vivo outcomes after hydrogel injection and IL-10 delivery. Eight groups were investigated (7, 21, 35 days, n = 4): healthy, sham, healthy injected with mouse serum albumin (MSA), healthy + hydrogel, UUO, UUO + IL-10, UUO + hydrogel, UUO + hydrogel/IL-10. 15 µL of IL-10, hydrogel, or hydrogel/IL-10 was injected under the renal capsule 3 days after the UUO. Immunohistochemistry (IHC) was performed on paraffin sections to identify macrophages and apoptotic cells and trichrome staining was used to evaluate fibrosis. There were no significant differences in inflammatory markers between all control groups. With hydrogel delivery, macrophage infiltration and apoptosis were significantly reduced at days 21 and 35 compared to untreated animals. By day 35, IL-10 delivery via hydrogel reduced macrophage infiltration and apoptosis more than IL-10 injection alone. Fibrosis was decreased by day 35 in all treatment groups. This work supports the use of hydrogel delivery of IL-10 to treat chronic kidney disease.
Assuntos
Hidrogéis , Imunoterapia , Nefropatias/terapia , Animais , Apoptose , Modelos Animais de Doenças , Interleucina-10/metabolismo , Nefropatias/metabolismo , Nefropatias/patologia , Macrófagos/citologia , Camundongos , Camundongos Endogâmicos BALB CRESUMO
Shear-thinning hydrogels are useful in numerous applications, including as injectable carriers that act as scaffolds to support cell and drug therapies. Here, we describe the engineering of a self-assembling Dock-and-Lock (DnL) system that forms injectable shear-thinning hydrogels using molecular recognition interactions that also possess photo-triggerable secondary crosslinks. These DnL hydrogels are fabricated from peptide-modified hyaluronic acid (HA) and polypeptide precursors, can self-heal immediately after shear induced flow, are cytocompatible, and can be stabilized through light-initiated radical polymerization of methacrylate functional groups to tune gel mechanics and erosion kinetics. Secondary crosslinked hydrogels retain self-adhesive properties and exhibit cooperative physical and chemical crosslinks with moduli as high as â¼10 times larger than moduli of gels based on physical crosslinking alone. The extent of reaction and change in properties are dependent on whether the methacrylate is incorporated either at the terminus of the peptide or directly to the HA backbone. Additionally, the gel erosion can be monitored through an incorporated fluorophore and physical-chemical gels remain intact in solution over months, whereas physical gels that are not covalently crosslinked erode completely within days. Mesenchymal stem cells exhibit increased viability when cultured in physical- chemical gels, compared with those cultured in gels based on physical crosslinks alone. The physical properties of these DnL gels may be additionally tuned by adjusting component compositions, which allows DnL gels with a wide range of physical properties to be constructed for use.