Two-Photon Microscopy Analysis of Gold Nanoparticle Uptake in 3D Cell Spheroids.
ABSTRACT: Nanomaterials can be synthesized from a wide range of material systems in numerous morphologies, creating an extremely diverse portfolio. As result of this tunability, these materials are emerging as a new class of nanotherapeutics and imaging agents. One particularly interesting nanomaterial is the gold nanoparticle. Due to its inherent biocompatibility and tunable photothermal behavior, it has made a rapid transition from the lab setting to in vivo testing. In most nanotherapeutic applications, the efficacy of the agent is directly related to the target of interest. However, the optimization of the AuNP size and shape for efficacy in vitro, prior to testing in in vivo models of a disease, has been largely limited to two dimensional monolayers of cells. Two dimensional cell cultures are unable to reproduce conditions experienced by AuNP in the body. In this article, we systematically investigate the effect of different properties of AuNP on the penetration depth into 3D cell spheroids using two-photon microscopy. The 3D spheroids are formed from the HCT116 cell line, a colorectal carcinoma cell line. In addition to studying different sizes and shapes of AuNPs, we also study the effect of an oligo surface chemistry. There is a significant difference between AuNP uptake profiles in the 2D monolayers of cells as compared to the 3D cell spheroids. Additionally, the range of sizes and shapes studied here also exhibit marked differences in uptake penetration depth and efficacy. Finally, our results demonstrate that two-photon microscopy enables quantitative AuNP localization and concentration data to be obtained at the single spheroid level without fluorescent labeling of the AuNP, thus, providing a viable technique for large scale screening of AuNP properties in 3D cell spheroids as compared to tedious and time consuming techniques like electron microscopy.
Project description:Current cancer therapies are frequently ineffective and associated with severe side effects and with acquired cancer drug resistance. The development of effective therapies has been hampered by poor correlations between pre-clinical and clinical outcomes. Cancer cell-derived spheroids are three-dimensional (3D) structures that mimic layers of tumors in terms of oxygen and nutrient and drug resistance gradients. Gold nanoparticles (AuNP) are promising therapeutic agents which permit diminishing the emergence of secondary effects and increase therapeutic efficacy. In this work, 3D spheroids of Doxorubicin (Dox)-sensitive and -resistant colorectal carcinoma cell lines (HCT116 and HCT116-DoxR, respectively) were used to infer the potential of the combination of chemotherapy and Au-nanoparticle photothermy in the visible (green laser of 532 nm) to tackle drug resistance in cancer cells. Cell viability analysis of 3D tumor spheroids suggested that AuNPs induce cell death in the deeper layers of spheroids, further potentiated by laser irradiation. The penetration of Dox and earlier spheroid disaggregation is potentiated in combinatorial therapy with Dox, AuNP functionalized with polyethylene glycol (AuNP@PEG) and irradiation. The time point of Dox administration and irradiation showed to be important for spheroids destabilization. In HCT116-sensitive spheroids, pre-irradiation induced earlier disintegration of the 3D structure, while in HCT116 Dox-resistant spheroids, the loss of spheroid stability occurred almost instantly in post-irradiated spheroids, even with lower Dox concentrations. These results point towards the application of new strategies for cancer therapeutics, reducing side effects and resistance acquisition.
Project description:Tumor microenvironments present significant barriers to penetration by antibodies and immunoconjugates. Tumor microenvironments, however, are difficult to study in vitro. Cells cultured as monolayers exhibit less resistance to therapy than those grown in vivo and an alternative research model more representative of the in vivo tumor is more desirable. SS1P is an immunotoxin composed of the Fv portion of a mesothelin-specific antibody fused to a bacterial toxin that is presently undergoing clinical trials in mesothelioma.Here, we examined how the tumor microenvironment affects the penetration and killing activity of SS1P in a new three-dimensional (3D) spheroid model cultured in vitro using the human mesothelioma cell line (NCI-H226) and two primary cell lines isolated from the ascites of malignant mesothelioma patients. Mesothelioma cells grown as monolayers or as spheroids expressed comparable levels of mesothelin; however, spheroids were at least 100 times less affected by SS1P. To understand this disparity in cytotoxicity, we made fluorescence-labeled SS1P molecules and used confocal microscopy to examine the time course of SS1P penetration within spheroids. The penetration was limited after 4 hours. Interestingly, we found a significant increase in the number of tight junctions in the core area of spheroids by electron microscopy. Expression of E-Cadherin, a protein involved in the assembly and sealing of tight junctions and highly expressed in malignant mesothelioma, was found significantly increased in spheroids as compared to monolayers. Moreover, we found that siRNA silencing and antibody inhibition targeting E-Cadherin could enhance SS1P immunotoxin therapy in vitro.This work is one of the first to investigate immunotoxins in 3D tumor spheroids in vitro. This initial description of an in vitro tumor model may offer a simple and more representative model of in vivo tumors and will allow for further investigations of the microenvironmental effects on drug penetration and tumor cell killing. We believe that the methods developed here may apply to the studies of other tumor-targeting antibodies and immunoconjugates in vitro.
Project description:Three-dimensional (3D) cell cultures allow the mimic of functions of living tissues andprovide key information encoded in tissue architecture. Considered the pivotal role of epithelial-tomesenchymaltransition (EMT) in carcinoma progression, including prostate cancer (PCa), weaimed at investigating the effect of the 3D arrangement on the expression of some key markers ofEMT in cultured human prostate cancer (PCa) cells, to better understand PCa cell behavior. PC3 andDU145 PCa cells were cultured in RPMI cell culture medium either in 2D-monolayers or in 3Dspheroids.The main EMT markers E-cadherin, N-cadherin, α-smooth muscle actin (αSMA),vimentin, Snail, Slug, Twist and Zeb1 were evaluated by confocal microscopy, real-time PCR andWestern blot. Confocal microscopy revealed that E-cadherin was similarly expressed at the cellboundaries on the plasma membrane of PCa cells grown in 2D-monolayers, as well as in 3Dspheroids,but resulted up-regulated in 3D-spheroids, compared to 2D-monolayers, at the mRNAand protein level. Moreover, markers of the mesenchymal phenotype were expressed at very lowlevels in 3D-spheroids, suggesting important differences in the phenotype of PCa cells grown in 3Dspheroidsor in 2D-monolayers. Considered as a whole, our findings contribute to a clarification ofthe role of EMT in PCa and confirm that a 3D cell culture model could provide deeper insight intothe understanding of the biology of PCa.
Project description:Nanomaterials have been extensively investigated for cancer drug delivery and imaging applications. Nanoparticles that show promise in two-dimensional cell culture systems often fail in more complex environments, possibly due to the lack of penetration in dense, three-dimensional structures. Multicellular tumor spheroids are an emerging model system to investigate interactions of nanoparticles with 3D in vitro cell culture environments. Using the intrinsic near-infrared emission of semiconducting carbon nanotubes to optically reconstruct their localization within a three-dimensional volume, we resolved the relative permeability of two different multicellular tumor spheroids. Nanotube photoluminescence revealed that nanotubes rapidly internalized into MCF-7 breast cancer cell-derived spheroids, whereas they exhibited little penetration into spheroids derived from SK-136, a cell line that we developed from murine liver cancer. Characterization of the spheroids by electron microscopy and immunohistochemistry revealed large differences in the extracellular matrix and interstitial spacing, which correlated directly with nanotube penetration. This platform portends a new approach to characterize the permeability of living multicellular environments.
Project description:To enhance the accumulation and penetration of nanomedicines in tumor tissue, we developed and evaluated the biological properties of matrix metalloproteinase 2 (MMP-2)-responsive N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer drugs and tumor-penetrating peptide conjugates (P-DOX-PLGLAG-iRGD). Two different spacers were used in the design: a lysosomally (cathepsin B) cleavable tetrapeptide GFLG spacer conjugated doxorubicin (DOX) to HPMA copolymer, and an MMP-2-degradable linker (PLGLAG) connected tumor-homing and -penetrating cyclic peptide iRGD to HPMA copolymer. The accumulation of DOX in P-DOX-PLGLAG-iRGD-treated monolayer (2D) and multilayer (3D) DU-145 prostate cancer cells was higher than that of control groups (P-DOX and P-DOX + iRGD). The cell cycle arrest analysis and cytotoxicity data demonstrated that P-DOX-PLGLAG-iRGD produced a higher G2/M arrest and possessed stronger cytotoxicity against DU-145 cells than P-DOX + iRGD or P-DOX, which was consistent with the drug uptake results. Similarly, P-DOX-PLGLAG-iRGD demonstrated the highest penetration ability in 3D multicellular DU-145 tumor cell spheroids. The results indicate that covalent conjugation of iRGD via MMP-2-sensitive bonds enhances accumulation and penetration of nanomedicines into tumor cell monolayers and spheroids.
Project description:Introduction:Nanoparticles (NPs) are used in numerous products in technical fields and biomedicine; their potential adverse effects have to be considered in order to achieve safe applications. Besides their distribution in tissues, organs, and cellular localization, their impact and penetration during the process of tissue formation occurring in vivo during liver regeneration are critical steps for establishment of safe nanomaterials. Materials and methods:In this study, 3D cell culture of human hepatocarcinoma cells (HepG2) was used to generate cellular spheroids, serving as in vitro liver microtissues. In order to determine their differential distribution and penetration depth in HepG2 spheroids, SiO2 NPs were applied either during or after spheroid formation. The NP penetration was comprehensively studied using confocal laser scanning microscopy and scanning electron microscopy. Results:Spheroids were exposed to 100 µg mL-1 SiO2 NPs either at the beginning of spheroid formation, or during or after formation of spheroids. Microscopy analyses revealed that NP penetration into the spheroid is limited. During and after spheroid formation, SiO2 NPs penetrated about 20 µm into the spheroids, corresponding to about three cell layers. In contrast, because of the addition of SiO2 NPs simultaneously to cell seeding, NP agglomerates were located also in the spheroid center. Application of SiO2 NPs during the process of spheroid formation had no impact on final spheroid size. Conclusion:Understanding the distribution of NPs in tissues is essential for biomedical applications. The obtained results indicate that NPs show only limited penetration into already formed tissue, which is probably caused by the alteration of the tissue structure and cell packing density during the process of spheroid formation.
Project description:The aim of our study was to evaluate the influence of low-intensity pulsed US on the delivery of doxorubicin (DOX) into MDA-MB-231 triple-negative breast cancer and A549 non-small cell lung cancer cell 2D and 3D cultures. US with pulse repetition frequency of 10 Hz and 1 MHz center frequency was generated with peak negative pressure of 0.5 MPa and 50% duty cycle. SonoVue microbubbles were used. Spheroids were formed using 3D Bioprinting method. DOX delivery in 2D and 3D cultures was assessed using fluorescence microscopy. US without the addition of microbubbles did not enhance the penetration of DOX into monolayer-cultured cells and tumor spheroids. In the presence of microbubbles US improved the delivery of DOX into the edge end middle zones of A549 and MDA-MB-231 spheroids. Application of low-intensity pulsed US in combination with microbubbles may be a promising approach to enhance the delivery of DOX into tumor spheroids.
Project description:The impact of electrostatic attraction on the uptake of gold nanoparticles (AuNPs) into positively charged strong poly-[2-(Methacryloyloxy) ethyl] trimethylammonium chloride (PMETAC) polyelectrolyte brushes was investigated. In this work, PMETAC brushes were synthesized via surface-initiated atom transfer radical polymerization (Si-ATRP). PMETAC/AuNP composite materials were prepared by incubation of the polymer brush coated samples into 3-mercaptopropionic acid-capped AuNP (5 nm in diameter) suspension. The electrostatic interactions were tuned by changing the surface charge of the AuNPs through variations in pH value, while the charge of the PMETAC brush was not affected. Atomic-force microscopy (AFM), ellipsometry, UV/Vis spectroscopy, gravimetric analysis and transmission electron microscopy (TEM) were employed to study the loading and penetration into the polymer brush. The results show that the number density of attached AuNPs depends on the pH value and increases with increasing pH value. There is also strong evidence that the particle assembly is dependent on the pH value of the AuNP suspension. Incubation of PMETAC brushes in AuNP suspension at pH 4 led to the formation of a surface layer on top of the brush (2D assembly) due to sterical hindrance of the clustered AuNPs, while incubation in AuNP suspension at pH 8 led to deeper particle penetration into the brush (3D assembly). The straightforward control of particle uptake and assembly by tuning the charge density of the nanoparticle surface is a valuable tool for the development of materials for colorimetric sensor applications.
Project description:In this work, three-dimensional (3D) spheroid cultures of human adipose-derived mesenchymal stem cells (hAD-MSCs), with tissue-mimetic morphology through well developed cell-cell and cell-matrix interactions and distinct diffusion/transport characteristics, were assessed for dose-dependent toxic effects of red-emitting CdTe/CdS/ZnS quantum dots (Qdots). Morphological investigations and time-resolved microscopy analysis in addition to cell metabolic activity studies revealed that 3D spheroid cultures are more resistant to Qdot-induced cytotoxicity in comparison to conventional 2D cultures. The obtained results suggest the presence of two distinct cell populations in 2D cultures with different sensitivity to Qdots, however that effect wasn't observed in 3D spheroids. Our investigations were aimed to improve the prediction of nanotoxicity of Qdot on tissue-level and provide the essential screening steps prior to any in vivo application. Moreover, penetration ability of highly fluorescent Qdots to densely-packed spheroids will fortify the biological application of developed Qdots in tissue-like structures.
Project description:Purpose:The purpose of our study was to evaluate the influence of two PPIs (omeprazole (OME) and lansoprazole (LANSO)) on weakly basic anticancer drug doxorubicin (DOX) and pegylated liposomal doxorubicin (PLD) delivery to monolayer-cultured 4T1 murine breast cancer cells and tumor spheroids. Methods:The effect of PPIs on cell viability was evaluated by MTT assay. 3D cell cultures (spheroids) were formed using 3D bioprinting method. DOX and PLD penetration into cancer cells and spheroids at pH 6.0 and 7.4 was assessed using fluorescence microscopy. Results:Both OME and LANSO did not reduce the viability of 4T1 cells at 100 ?M and lower concentrations, and therefore, in further experiments, 100 ?M of PPIs was used. At pH 7.4, both tested PPIs did not enhance DOX (5 µM) and PLD (concentration corresponding to 5 µM DOX) delivery into 2D cell cultures. However, in acidic conditions, both PPIs increased the amount of drug in cancer cells and their nucleus. At physiological pH they were not effective at improving DOX delivery into spheroids, but after 2 hrs of incubation, OME slightly increased PLD delivery into edge and middle zones. At pH 6.0, both tested PPIs significantly enhanced DOX and PLD transport into spheroids, but the positive effect on delivery was observed only within the first 4 hrs of incubation. Conclusion:Both OME and LANSO increased DOX and PLD penetration into monolayer-cultured cells at acidic conditions but did not show a positive effect on drug delivery at physiological pH. Also, pretreatment with tested PPIs slightly increased DOX and PLD delivery in the edge and middle zones of tumor spheroids. Thus, OME and LANSO are promising transport modulators of weakly basic drugs.