RESUMO
Biocompatible nanoparticles as drug carriers can improve the therapeutic efficiency of hydrophobic drugs. However, the synthesis of biocompatible and biodegradable polymeric nanoparticles can be time-consuming and often involves toxic solvents. Here, a simple method for protein-based stable drug-loaded particles with a narrow polydispersity is introduced. In this process, lysozyme is mixed with hydrophobic drugs (curcumin, ellipticine, and dasatinib) and fructose to prepare lysozyme-based drug particles of around 150 nm in size. Fructose is mixed with the drug to generate nanoparticles that serve as templates for the lysozyme coating. The effect of lysozyme on the physicochemical properties of these nanoparticles is studied by transmission electron microscopy (TEM) and scattering techniques (e.g., dynamic light scattering (DLS) and small-angle X-ray scattering (SAXS)). We observed that lysozyme significantly stabilized the curcumin fructose particles for 7 days. Moreover, additional drugs, such as ellipticine and dasatinib, can be loaded to form dual-drug particles with narrow polydispersity and spherical morphology. The results also reveal that lysozyme dual ellipticine/dasatinib curcumin particles enhance the cytotoxicity and uptake on MCF-7 cells, RAW 264.7 cells, and U-87 MG cells due to the larger and rigid hydrophobic core. In summary, lysozyme in combination with fructose and curcumin can serve as a powerful combination to form protein-based stable particles for the delivery of hydrophobic drugs.
Assuntos
Curcumina , Dasatinibe , Portadores de Fármacos , Elipticinas , Muramidase , Nanopartículas , Muramidase/química , Muramidase/metabolismo , Nanopartículas/química , Curcumina/química , Curcumina/farmacologia , Animais , Humanos , Camundongos , Portadores de Fármacos/química , Dasatinibe/química , Dasatinibe/farmacologia , Elipticinas/química , Elipticinas/farmacologia , Células RAW 264.7 , Células MCF-7 , Tamanho da Partícula , Frutose/química , Interações Hidrofóbicas e Hidrofílicas , Sobrevivência Celular/efeitos dos fármacos , Linhagem Celular TumoralRESUMO
The field of single-chain nanoparticles (SCNPs) continues to mature, and an increasing range of reports have emerged that explore the application of these small nanoparticles. A key application for SCNPs is in the field of drug delivery, and recent work suggests that SCNPs can be readily internalized by cells. However, limited attention has been directed to the delivery of small-molecule drugs using SCNPs. Moreover, studies on the physicochemical effects of drug loading on SCNP performance is so far missing, despite the accepted view that such small nanoparticles should be significantly affected by the drug loading content. To address this gap, we prepared a library of SCNPs bearing different amounts of a covalently conjugated therapeutic drug-sulfasalazine (SSZ). We evaluated the impact of the conjugated drug loading on both the synthesis and biological activity of SCNPs on pancreatic cancer cells (AsPC-1). Our results reveal that covalent drug conjugation to the side chains of the SCNP polymer precursor interferes with chain collapse and cross-linking, which demands optimization of reaction conditions to reach high degrees of cross-linking efficiencies. Small-angle neutron scattering and diffusion-ordered spectroscopy nuclear magnetic resonance (DOSY NMR) analyses reveal that SCNPs with a higher drug loading display larger sizes and looser structures, as well as increased hydrophobicity associated with a higher SSZ content. Increased SSZ loading led to reduced cellular uptake when assessed in vitro, whereby SCNP aggregation on the surface of AsPC-1 cells led to reduced toxicity. This work highlights the effects of drug loading on the drug delivery efficiency and biological behavior of SCNPs.
Assuntos
Nanopartículas , Nanopartículas/química , Sistemas de Liberação de Medicamentos/métodos , Polímeros/química , Preparações FarmacêuticasRESUMO
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) drives apoptosis selectively in cancer cells by clustering death receptors (DR4 and DR5). While it has excellent in vitro selectivity and toxicity, the TRAIL protein has a very low circulation half-life in vivo, which has hampered clinical development. Here, we developed core-cross-linked micelles that present multiple copies of a TRAIL-mimicking peptide at its surface. These micelles successfully induce apoptosis in a colon cancer cell line (COLO205) via DR4/5 clustering. Micelles with a peptide density of 15% (roughly 1 peptide/45 nm2) displayed the strongest activity with an IC50 value of 0.8 µM (relative to peptide), demonstrating that the precise spatial arrangement of ligands imparted by a protein such as a TRAIL may not be necessary for DR4/5/signaling and that a statistical network of monomeric ligands may suffice. As micelles have long circulation half-lives, we propose that this could provide a potential alternative drug to TRAIL and stimulate the use of micelles in other membrane receptor clustering networks.
Assuntos
Proteínas Reguladoras de Apoptose , Neoplasias do Colo , Humanos , Proteínas Reguladoras de Apoptose/metabolismo , Proteínas Reguladoras de Apoptose/farmacologia , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Micelas , Ligantes , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Linhagem Celular Tumoral , Apoptose , Fator de Necrose Tumoral alfa/metabolismo , Neoplasias do Colo/tratamento farmacológico , Peptídeos/farmacologia , Peptídeos/metabolismo , Proteínas de TransporteRESUMO
While the effects of nanoparticle properties such as shape and size on cellular uptake are widely studied, influences exerted by drug loading have so far been ignored. In this work, nanocellulose (NC) coated by Passerini reaction with poly(2-hydroxy ethyl acrylate) (PHEA-g-NC) was loaded with various amounts of ellipticine (EPT) by electrostatic interactions. The drug-loading content was determined by UV-vis spectroscopy to range between 1.68 and 8.07 wt %. Dynamic light scattering and small-angle neutron scattering revealed an increased dehydration of the polymer shell with increasing drug-loading content, which led to higher protein adsorption and more aggregation. The nanoparticle with the highest drug-loading content, NC-EPT8.0, displayed reduced cellular uptake in U87MG glioma cells and MRC-5 fibroblasts. This also translated into reduced toxicity in these cell lines as well as the breast cancer MCF-7 and the macrophage RAW264.7 cell lines. Additionally, the toxicity in U87MG cancer spheroids was unfavorable. The nanoparticle with the best performance was found to have intermediate drug-loading content where the cellular uptake was adequately high while each nanoparticle was able to deliver a sufficiently toxic amount into the cells. Medium drug loading did not hinder uptake into cells while maintaining sufficiently toxic drug concentrations. It was concluded that while striving for a high drug-loading content is appropriate when designing clinically relevant nanoparticles, it needs to be considered that the drug can cause changes in the physicochemical properties of the nanoparticles that might cause unfavorable effects.
Assuntos
Neoplasias da Mama , Nanopartículas , Humanos , Feminino , Polímeros/química , Portadores de Fármacos/química , Linhagem Celular , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Macrófagos , Nanopartículas/químicaRESUMO
Margination describes the movement of particles toward the endothelial wall within blood vessels. While there have been several studies tracking the margination of spherical particles in blood, the behavior of anisotropic particle shapes is not well described. In this study 2D platelet particles which possess many attractive qualities for use as a drug delivery system, with their high surface area allowing for increased surface binding activity, were directly monitored and margination quantified. The margination propensity of 1 and 2 µm 2D platelet particles was contrasted to that of 2 µm spherical particles at apparent wall shear rates (WSRs) of 50, 100, and 200 s-1 by both directly tracking labeled particles using fluorescent microscopy as well as using small-angle X-ray scattering (SAXS). For fluorescence studies, margination was quantified using the margination parameter M, which describes the number of particles found closest to the walls of a microfluidic device, with an M-value of 0.2 indicating no margination. Increased margination was seen in 2D platelet particles when compared to spherical particles tested at all flow rates, with M-values of 0.39 and 0.31 seen for 1 and 2 µm 2D platelet particles, respectively, while 2 µm spherical particles had an M-value of 0.21. Similarly, margination was observed qualitatively using SAXS, with increased scattering seen for platelet particles near the microfluidic channel wall. For all particles, increased margination was seen at increasing shear rates.
Assuntos
Plaquetas , Sistemas de Liberação de Medicamentos , Espalhamento a Baixo Ângulo , Difração de Raios XRESUMO
Understanding cellular uptake and particle trafficking within the cells is essential for targeted drug delivery applications. Existing studies reveal that the geometrical aspects of nanocarriers, for example, shape and size, determine their cell uptake and sub-cellular transport pathways. However, considerable efforts have been directed toward understanding the cell uptake mechanism and trafficking of spherical particles. Detailed analysis on the uptake mechanism and downstream intracellular processing of non-spherical particles remains elusive. Here, we used polymeric two-dimensional platelets based on poly(ε-caprolactone) (PCL) prepared by living crystallization-driven self-assembly as a platform to investigate the cell uptake and intracellular transport of non-spherical particles in vitro. PCL is known to degrade only slowly, and these platelets were still stable after 2 days of incubation in artificial lysosomal media. Upon cell uptake, the platelets were transported through an endo/lysosomal pathway and were found to degrade completely in the lysosome at the end of the cell uptake cycle. We observed a morphological transformation of the lysosomes, which correlates with the stages of platelet degradation in the lysosome. Overall, we found an accelerated degradation of PCL, which was likely caused by mechanical forces inside the highly stretched endosomes.
Assuntos
Poliésteres , Polietilenoglicóis , Lisossomos , MacrófagosRESUMO
Tumor targeting has revolutionized cancer research, especially active cellular targeting of nanoparticles, where they are specifically homed to the pathological site to deliver the therapeutics. This strategy, which involves the utilization of affinity ligands on the surface of the nanocarriers, minimizes the nonspecific uptake of nanocarriers and the subsequent harmful side effects in healthy cells. Estrone, one of the mammalian estrogens, has affinity for estrogen receptors (ERα), which are overexpressed in hormone-responsive breast cancers. Despite holding promise, the potential of estrone in active targeting of nanoparticles has barely been explored. Herein, we developed an estrone-appended polyion complex (PIC) micelle to deliver melittin, a cytotoxic peptide, to breast cancer cells. Amino functionalization of estrone was performed to conjugate estrone to the diblock polymer synthesized by reversible addition-fragmentation chain-transfer (RAFT) polymerization. Estrone-conjugated poly(ethylene glycol) methyl ether methacrylate-b-poly tert-butyl methacrylate (POEGMEMA-PtBuMA) could complex with melittin to form PIC micelles of size around 60 nm ensuing from the electrostatic interaction of the deprotected polymer and melittin in aqueous media. Poly(ethylene glycol) methyl ether acrylate-b-poly acrylic acid (POEGMEA-PAA) was also later incorporated to afford PIC micelles that could exhibit similar cytotoxicity to free melittin in the cytotoxicity studies. The estrone-attached PIC micelles exhibited improved cytotoxicity in two-dimensional (2D) and three-dimensional (3D) cellular models of MCF-7 cells. Cross-linking of the PIC micelles was also performed to improve the stability of the micelles and prevent melittin degradation from enzymatic attack. Flow cytometry demonstrated an enhanced cellular uptake greater than sixfold with the estrone-conjugated PIC micelles, thereby establishing a profound difference in the targeting efficacy of the PIC micelles between MCF-7 and MDA-MB-231 cells. Furthermore, the distribution of the PIC micelles in the spheroids was revealed by light sheet microscopy. The results demonstrate the potential of estrone-anchored PIC micelles for targeted delivery of therapeutics to hormone-responsive breast cancer cells.
Assuntos
Neoplasias da Mama , Micelas , Animais , Neoplasias da Mama/tratamento farmacológico , Portadores de Fármacos , Estrona , Feminino , Humanos , Meliteno , Polietilenoglicóis , PolímerosRESUMO
Nanocellulose is an excellent carrier to deliver drugs, as the material is biocompatible and has a desirable non-spherical shape. However, nanocellulose displays low solubility in aqueous solution and needs to be modified with water-soluble polymers in order to achieve high colloidal stability. In this study, (2,2,6,6-tetramethylpiperidin-1-yl)oxyl or (2,2,6,6-tetramethylpiperidin-1-yl)oxidanyl (TEMPO)-oxidized cellulose nanofibers bearing carboxylic acid moieties (TOCNs) are modified by nitrile imine-mediated tetrazole/carboxylic acid ligation. The advantage of this reaction is that TOCNs do not need to be modified further and the polymer with tetrazole end-functionalities can be directly clicked onto the TOCNs forming fluorescent functional groups. Poly(2-hydroxyethyl acrylate) with a tetrazole end-functionality is prepared using RAFT polymerization. The polymer is mixed with TOCNs and after irradiation at λ = 326 nm for 10 h, fluorescent pHEA-g-TOCNs are obtained. The polymer-grafted nanocellulose is found to disperse well in water and has only limited albumin binding. The uptake of these nanoparticles by MCF-7 breast cancer cell lines can now be monitored by fluorescent microscopy without further modification. Excess negatively charged carboxylic groups of TOCNs allow doxorubicin loading by electrostatic interactions at various drug-loading capacities. Higher drug loading is more efficient in inhibiting the cell proliferation, highlighting the effect of drug loading on toxicity.
Assuntos
Celulose/química , Portadores de Fármacos/química , Nanofibras/química , Ácidos Carboxílicos/química , Proliferação de Células/efeitos dos fármacos , Óxidos N-Cíclicos/química , Doxorrubicina/química , Doxorrubicina/farmacologia , Humanos , Células MCF-7 , Microscopia de Fluorescência , Tetrazóis/química , Raios UltravioletaRESUMO
Curcumin, a component in spice turmeric, is renowned to possess anti-cancer therapeutic potential. However, low aqueous solubility and instability of curcumin which subsequently affects its bioavailability pose as major impediments in its translation to clinical application. In this regard, we focused on conjugating hydrophobic curcumin to the hydrophilic backbone of dextran via succinic acid spacer to design a pro-drug. The structural confirmation of the conjugates was carried out using FTIR and (1)H NMR spectroscopy. Critical micelle measurement affirmed the micelle formation of the pro-drug in aqueous media. The size distribution and zeta potential of the curcumin-dextran (Cur-Dex) micelles were determined using dynamic light scattering technique. The micellar architecture bestowed curcumin negligible susceptibility to degradation under physiological conditions along with enhanced aqueous solubility. Biocompatibility of the micelles was proved by the blood component aggregation and plasma protein interaction studies. In vitro release studies demonstrated the pH sensitivity release of curcumin which is conducive to the tumour micro environment. Profound cytotoxic effects of Cur-Dex micelles in C6 glioma cells were observed from MTT and Live/Dead assay experiments. Moreover, enhanced cellular internalization of the Cur-Dex micelles compared to free curcumin in the cancer cells was revealed by fluorescence microscopy. Our study focuses on the feasibility of Cur-Dex micelles to be extrapolated as promising candidates for safe and efficient cancer therapy.
Assuntos
Antineoplásicos/química , Curcumina/análogos & derivados , Dextranos/química , Micelas , Pró-Fármacos/química , Antineoplásicos/efeitos adversos , Células Sanguíneas/efeitos dos fármacos , Linhagem Celular Tumoral , Células Cultivadas , Hemólise , Humanos , Concentração de Íons de Hidrogênio , Pró-Fármacos/efeitos adversosRESUMO
Clinical application of curcumin has been limited due to poor aqueous solubility and consequently minimal systemic bioavailability. We investigated the preparation of curcumin-loaded micelles based on amphiphilic Pluronic/Polycaprolactone (Pluronic/PCL) block copolymer, which proved to be efficient in enhancing curcumin's aqueous solubility. Curcumin-loaded micelles of size below 200 nm was characterized by dynamic light scattering and transmission electron microscopy. The critical micelle concentration (CMC) of the amphiphilic polymer was determined using pyrene as a fluorescent probe. Hemolysis and aggregation studies were investigated to evaluate the blood compatibility of the micelles. Sodium dodecyl sulphate polyacrylamide gel electrophoresis was performed to study the stability of the micelles toward plasma proteins. In vitro cytotoxicity and cellular uptake of the curcumin-loaded micelles were demonstrated in colorectal adenocarcinoma (Caco2) cells. The results indicated that Pluronic/PCL micelles could be a promising candidate for curcumin delivery to cancer cells.