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Tumor spheroids represent valuable in vitro models for studying cancer biology and evaluating therapeutic strategies. In this study, we investigated the impact of varying lengths of DNA-modified cell surfaces on spheroid formation, cellular adhesion molecule expression, and hypoxia levels within 4T1 mouse breast cancer spheroids. Through a series of experiments, we demonstrated that modifying cell surfaces with biotinylated DNA strands of different lengths facilitated spheroid formation without significantly altering the expression of fibronectin and e-cadherin, key cellular adhesion molecules. However, our findings revealed a notable influence of DNA length on hypoxia levels within the spheroids. As DNA length increased, hypoxia levels decreased, indicating enhanced intercellular spacing and porosity within the spheroid structure. These results contribute to a better understanding of how DNA modification of cell surfaces can modulate spheroid architecture and microenvironmental conditions. Such insights may have implications for developing therapeutic interventions targeting the tumor microenvironment to improve cancer treatment efficacy.
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The role of stromal and immune cells in transforming the tumor microenvironment is a key consideration in understanding tumor cell behavior and anticancer drug development. To better model these systems in vitro, 3D coculture tumor spheroids have been engineered using a variety of techniques including centrifugation to microwells, hanging drop, low adhesion cultures, and culture of cells in a microfluidic platform. Aside from using bioprinting, however, it has remained more challenging to direct the spatial organization of heterotypic cells in standalone 3D spheroids. To address this, we present an in vitro 3D coculture tumor model where we modulated the interactions between cancer cells and fibroblasts through DNA hybridization. When native heterotypic cells are simply mixed, the cell aggregates typically show cell sorting behavior to form phase separated structures composed of single cell types. In this work, we demonstrate that when MDA-MB-468 breast cancer and NIH/3T3 fibroblasts are directed to associate via complementary DNA, a uniform distribution of the two cell types within a single spheroid was observed. In contrast, in the absence of specific DNA interactions between the cancer cells and fibroblasts, individual clusters of the NIH/3T3 cells formed in each spheroid due to cell sorting. To better understand the effect of heterotypic cell organization on either cell-cell contacts or matrix protein production, the spheroids were further stained with anti-E-cadherin and antifibronectin antibodies. While the amounts of E-cadherin appeared to be similar between the spheroids, a significantly higher amount of fibronectin secretion was observed in the coculture spheroids with uniform mixing of two cell types. This result showed that different heterotypic cell distributions within 3D architecture can influence the ECM protein production that can again alter the properties of the tumor or tumor microenvironment. The present study thus describes the use of DNA templating to direct the organization of cells in coculture spheroids, which can provide mechanistic biological insight into how heterotypic distribution in tumor spheroids can influence tumor progression, metastasis, and drug resistance.
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Neoplasias da Mama , Esferoides Celulares , Camundongos , Animais , Humanos , Feminino , Técnicas de Cocultura , Esferoides Celulares/metabolismo , Caderinas , DNA , Microambiente TumoralRESUMO
Hydrophobically-modified silica-coated gold nanorods are presented here as multifunctional theranostic agents. A single modification both increases two-photon fluorescence and promotes cavitation-based acoustic signal for imaging. A two-fold greater release of small molecule drugs was observed under ultrasound-mediated conditions as compared to passive release without ultrasound.
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Ouro , Dióxido de Silício , Liberação Controlada de FármacosRESUMO
In this work, we demonstrate that a photo-cross-linkable conjugate of upconverting nanoparticles and cytosine deaminase can catalyze prodrug conversion specifically at tumor sites in vivo. Non-covalent association of proteins and peptides with cellular surfaces leads to receptor-mediated endocytosis and catabolic degradation. Recently, we showed that covalent attachment of proteins such as affibodies to cell receptors yields extended expression on cell surfaces with preservation of protein function. To adapt this technology for in vivo applications, conjugates were prepared from upconverting nanoparticles and fusion proteins of affibody and cytosine deaminase enzyme (UC-ACD). The affibody allows covalent photo-cross-linking to epidermal growth factor receptors (EGFRs) overexpressed on Caco-2 human colorectal cancer cells under near-infrared (NIR) light. Once bound, the cytosine deaminase portion of the fusion protein converts the prodrug 5-fluorocytosine (5-FC) to the anticancer drug 5-fluorouracil (5-FU). NIR covalent photoconjugation of UC-ACD to Caco-2 cells showed 4-fold higher retention than observed with cells that were not irradiated in vitro. Next, athymic mice expressing Caco-2 tumors showed 5-fold greater UC-ACD accumulation in the tumors than either conjugates without the CD enzyme or UC-ACDs in the absence of NIR excitation. With oral administration of 5-FC prodrug, tumors with photoconjugated UC-ACD yielded 2-fold slower growth than control groups, and median mouse survival increased from 28 days to 35 days. These experiments demonstrate that enzyme-decorated nanoparticles can remain viable after a single covalent photoconjugation in vivo, which can in turn localize prodrug conversion to tumor sites for multiple weeks.
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Antineoplásicos , Nanopartículas , Pró-Fármacos , Humanos , Camundongos , Animais , Pró-Fármacos/farmacologia , Pró-Fármacos/metabolismo , Flucitosina/farmacologia , Flucitosina/metabolismo , Flucitosina/uso terapêutico , Citosina Desaminase/metabolismo , Células CACO-2 , Fluoruracila/metabolismo , Antineoplásicos/farmacologia , Camundongos Nus , Família de Proteínas EGF , Linhagem Celular TumoralRESUMO
Cellular quiescence is a reversible state of cell cycle arrest whereby cells are temporarily maintained in the nondividing phase. Inducing quiescence in cancer cells by targeting growth receptors is a treatment strategy to slow cell growth in certain aggressive tumors, which in turn increases the efficacy of treatments such as surgery or systemic chemotherapy. However, ligand interactions with cell receptors induce receptor-mediated endocytosis followed by proteolytic degradation, which limits the duration of cellular quiescence. Here, we report the effects of targeted covalent affibody photoconjugation to epidermal growth factor receptors (EGFR) on EGFR-positive MDA-MB-468 breast cancer cells. First, covalently conjugating affibodies to cells increased doubling time two-fold and reduced ERK activity by 30% as compared to cells treated with an FDA-approved anti-EGFR antibody Cetuximab, which binds to EGFR noncovalently. The distribution of cells in each phase of the cell cycle was determined, and cells conjugated with the affibody demonstrated an accumulation in the G1 phase, indicative of G1 cell cycle arrest. Finally, the proliferative capacity of the cells was determined by the incorporation of 5-ethynyl-2-deoxyuridine and Ki67 Elisa assay, which showed that the percentage of proliferative cells with photoconjugated affibody was half of that found for the untreated control.
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Morte Celular/efeitos dos fármacos , Receptores ErbB , Processos Fotoquímicos , Proteínas Recombinantes de Fusão , Neoplasias da Mama/metabolismo , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Receptores ErbB/química , Receptores ErbB/metabolismo , Feminino , Humanos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/farmacologiaRESUMO
3D culture is known to provide more faithful tissue models than 2D culture, and thus it is a valuable tool for in vitro evaluation of biological models. However, many cell lines are unable to form desired 3D spheroids by traditional methods because the naturally occurring cell-cell adhesion is too weak. Here, we present a method to produce 3D cell spheroids by using DNA-mediated assembly. We first demonstrate an Affinity Mediated Photoconjugation Approach (AMCP) to covalently modify cell receptors with affibody-streptavidin fusion proteins, where the affibody chemically crosslinks to cell expressed EGFR and the streptavidin is used to attach DNA strands. The DNA conjugated cells were then mixed with complementary DNA 'linker strands' to impart cell-cell interactions. When incubated in wells coated with non-adhesive polymers, cells formed dense spherical aggregates larger than 500 microns in diameter. Each of these studies was carried out using human breast cancer cells (MBA-MB-468), aneuploid human keratinocytes (HaCaT), and human colon cancer cells (Caco-2). Without either DNA on the cells or in solution as linkers, no cell spheroids were observed. After 96 h of incubation, the cultured DNA assembled spheroids were found to be mechanically stable enough to be handled easily for further analysis and confocal imaging. The findings suggest that the proposed DNA assembly method can be considered as an attractive strategy for assembling cells into stable spheroids.
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Comunicação Celular , Esferoides Celulares , Células CACO-2 , Adesão Celular , DNA , HumanosRESUMO
In this work we report the effect of incorporating conducting oligophenylenes and a cobaltocene-based redox mediator on photodriven electron transfer between thioglycolic acid (TGA) capped CdS nanorods (NR) and the native nitrogenase MoFe protein (MoFeP) by following the reduction of H+ to H2. First, we demonstrate that the addition of benzidine-a conductive diphenylene- to TGA-CdS and MoFeP increased catalytic activity by up to 3-fold as compared to CdS-MoFeP alone. In addition, in comparing the use of oligophenylenes composed of one (p-phenylenediamine), two (benzidine) or three (4,4''-diamino-p-terphenyl)phenylene groups, the largest gain in H2 was observed with the addition of benzidine and the lowest with phenylenediamine. As a comparison to the conductive oligophenylenes, a cobaltocene-based redox mediator was also tested with the TGA-CdS NRs and MoFeP. However, adding either cobaltocene diacid or diamine caused negligible gains in H2 production and at higher concentrations, caused a significant decrease. Agarose gel electrophoresis revealed little to no detectable interaction between benzidine and TGA-CdS but strong binding between cobaltocene and TGA-CdS. These results suggest that the tight binding of the cobaltocene mediator to CdS may hinder electron transfer between CdS and MoFe and cause the mediator to undergo continuous reduction/oxidation events at the surface of CdS.
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In this work, we report that gold nanorods coated with hydrophobically-modified mesoporous silica shells not only enhance photoacoustic (PA) signal over unmodified mesoporous silica coated gold nanorods, but that the relationship between PA amplitude and input laser fluence is strongly nonlinear. Mesoporous silica shells of ~14 nm thickness and with ~3 nm pores were grown on gold nanorods showing near infrared absorption. The silica was rendered hydrophobic with addition of dodecyltrichlorosilane, then re-suspended in aqueous media with a lipid monolayer. Analysis of the PA signal revealed not only an enhancement of PA signal compared to mesoporous silica coated gold nanorods at lower laser fluences, but also a nonlinear relationship between PA signal and laser fluence. We attribute each effect to the entrapment of solvent vapor in the mesopores: the vapor has both a larger expansion coefficient and thermal resistance than silica that enhances conversion to acoustic energy, and the hydrophobic porous surface is able to promote phase transition at the surface, leading to a nonlinear PA response even at fluences as low as 5 mJ cm-2. At 21 mJ cm-2, the highest laser fluence tested, the PA enhancement was >12-fold over mesoporous silica coated gold nanorods.
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This work reports the ability of hydrogel coatings to protect therapeutic proteins from cavitation-induced aggregation caused by mechanical stress. Here, we show that polyacrylamide hydrogel coatings on container surfaces suppress mechanical shock-induced cavitation and the associated aggregation of intravenous immunoglobulin (IVIg). First, crosslinked polyacrylamide hydrogels were grown on the surfaces of borosilicate glass vials. Treatment with ultrasound showed that these hydrogel surfaces suppressed cavitation events to levels below those found for unfunctionalized borosilicate glass. Next, IVIg solutions were loaded into these vials and subjected to tumbling, horizontal shaking, and drop testing. Aggregation was quantified by bisANS fluorescence staining and particle counting by flow imaging microscopy (FIM). In all cases, the presence of polyacrylamide hydrogels on the vial surfaces reduced the amount of IVIg aggregation and the number of particulates. In addition, the polyacrylamide appeared to have a protective effect that prevented additional aggregates from forming at extended tumbling times. Finally, drop test studies showed that the polyacrylamide coatings suppressed detectable cavitation. This work reveals how even a simple hydrogel vial coating can have a profound effect on stabilizing protein therapeutics.
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Imunoglobulinas Intravenosas , Agregados Proteicos , Hidrogéis , Estresse MecânicoRESUMO
This work reports the development of a mechanochemistry activated covalent conjugation (MACC) reaction that shows areas of interfacial failure in soft hydrogels. Hydrogels are prone to delamination from rigid substrates due to the competition between swelling and adhesion, which can lead to bonding failure in a mechanism similar to crack propagation in harder materials. In this work, reductive amination was shown to occur when a ketone-bearing fluorescein derivative was bonded to an amine-functionalized hydrogel, as both of these moieties were found to be necessary for covalent conjugation into the gel network. For thin, circular polyacrylamide hydrogels, wrinkle patterns and regions of subsequent delamination at the edge of the gel were found to be selectively tagged by the dye. This reaction was then used to explore the effect of gel properties on patterns of interfacial failure. As cross-linker loading increased, the propagation of the delamination front and the area fraction of delamination were both found to increase, as shown by fluorescence images of gels. Increasing the thickness of the gel increased the fraction of delaminated area but did not change its propagation toward the center of the gel. This MACC reaction shows how mechanochemical reactions can be used for fluorescence tagging without incorporating mechanophores into the polymer gel matrix.
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The cell membrane possesses an extensive library of proteins, carbohydrates, and lipids that control a significant portion of inter- and intracellular functions, including signaling, proliferation, migration, and adhesion, among others. Augmenting the cell surface composition would open possibilities for advances in therapy, tissue engineering, and probing fundamental cell processes. While genetic engineering has proven effective for many in vitro applications, these techniques result in irreversible changes to cells and are difficult to apply in vivo. Another approach is to instead attach exogenous functional groups to the cell membrane without changing the genetic nature of the cell. This review focuses on more recent approaches of nongenetic methods of cell surface modification through metabolic pathways, anchorage by hydrophobic interactions, and chemical conjugation. Benefits and drawbacks of each approach are considered, followed by a discussion of potential applications for nongenetic cell surface modification and an outlook on the future of the field.
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Proteínas de Membrana/química , Adesão Celular , Membrana Celular/química , Movimento Celular , Proliferação de Células , Interações Hidrofóbicas e Hidrofílicas , Transdução de SinaisRESUMO
Aggregation of therapeutic proteins can result from a number of stress conditions encountered during their manufacture, transportation, and storage. This work shows the effects of two interrelated sources of protein aggregation: the chemistry and structure of the surface of the container in which the protein is stored, and mechanical shocks that may result from handling of the formulation. How different mechanical stress conditions (dropping, tumbling, and agitation) and container surface passivation affect the stability of solutions of intravenous immunoglobulin are investigated. Application of mechanical shock causes cavitation to occur in the protein solution, followed by bubble collapse and the formation of high-velocity fluid microjets that impinged on container surfaces, leading to particle formation. Cavitation was observed after dropping of vials from heights as low as 5 cm, but polyethylene glycol (PEG) grafting provided temporary protection against drop-induced cavitation. PEG treatment of the vial surface reduced the formation of protein aggregates after repeated dropping events, most likely by reducing protein adsorption to container surfaces. These studies enable the development of new coatings and surface chemistries that can reduce the particulate formation induced by surface adsorption and/or mechanical shock.
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Embalagem de Medicamentos , Imunoglobulinas Intravenosas , Adsorção , Estresse MecânicoRESUMO
We illustrate how intermolecular interactions facilitate ATP-free electron transfer between either native or engineered MoFe protein (MoFeP) from nitrogenase and a CdS nanorod (NR) by following the reduction of H+ to H2. First, by varying the charge on the surface of the NR, we show the role of electrostatic interactions on MoFeP binding to the particle surface and subsequent H+ reduction. Next, the role of strong, semicovalent thiol-CdS interactions was tested using free cysteines on the MoFeP. By blocking free cysteines, we show that the presence of free thiols on the protein has little to no influence on CdS binding and resultant photocatalytic activity. We next studied methods to covalently bind the protein to CdS by modifying the free cysteines with dibenzocyclooctyne (DBCO) and reacting the CdS NRs capped with a mixture of negatively charged thioglycolic acid and thiol-PEG3-azide ligands. As compared to that of the unmodified proteins, a 32.2 ± 1.5% and 61.7 ± 2.1% increase in H2 production was observed from MoFeP and C-MoFeP, respectively. At last, to test the effect of both charge and covalent tethering, positively charged cysteamine/azide CdS NRs were reacted with DBCO-modified C-MoFeP, which showed little improvement over native C-MoFeP alone under irradiation. These results show the importance of both electrostatic associations between the NR and protein and covalently tethering the protein to the semiconductor surface for enhanced electron transfer and photodriven activity.
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In this work, we show that a prodrug enzyme covalently photoconjugated to live cell receptors survives endosomal proteolysis and retains its catalytic activity over multiple days. Here, a fusion protein was designed with both an antiepidermal growth factor receptor (EGFR) affibody and the prodrug enzyme cytosine deaminase, which can convert prodrug 5-fluorocytosine to the anticancer drug 5-fluorouracil. A benzophenone group was added at a site-specific mutation within the affibody, and the fusion protein was selectively photoconjugated to EGFR receptors expressed on membranes of MDA-MB-468 breast cancer cells. The fusion protein was next labeled with two dyes for tracking uptake: AlexaFluor 488 and pH-sensitive pHAb. Flow cytometry showed that fusion proteins photo-cross-linked to EGFR first underwent receptor-mediated endocytosis within 12 h, followed by recycling back to the cell membrane within 24 h. These findings were also confirmed by confocal microscopy. The unique cross-linking of the affibody-enzyme fusion proteins was utilized for two anticancer treatments. First, the covalent linking of the protein to the EGFR led to inhibition of ERK signaling over a two-day period, whereas conventional antibody therapy only led to 6 h of inhibition. Second, when the affibody-CodA fusion proteins were photo-cross-linked to EGFR overexpressed on MDA-MB-468 breast cancer cells, prodrug conversion was found even 48 h postincubation without any apparent decrease in cell killing, while without photo-cross-linking no cell killing was observed 8 h postincubation. These studies show that affinity-mediated covalent conjugation of the affibody-enzymes to cell receptors allows for prolonged expression on membranes and retained enzymatic activity without genetic engineering.
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Antineoplásicos/farmacologia , Citosina Desaminase/farmacologia , Receptores ErbB/antagonistas & inibidores , Flucitosina/farmacologia , Fluoruracila/farmacologia , Pró-Fármacos/farmacologia , Antineoplásicos/farmacocinética , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Citosina Desaminase/farmacocinética , Receptores ErbB/metabolismo , Feminino , Flucitosina/farmacocinética , Fluoruracila/farmacocinética , Humanos , Pró-Fármacos/farmacocinética , Inibidores de Proteínas Quinases/farmacocinética , Inibidores de Proteínas Quinases/farmacologia , Proteínas Recombinantes de Fusão/farmacocinética , Proteínas Recombinantes de Fusão/farmacologiaRESUMO
Here we report the efficacy of a nanoparticle-assisted high-intensity focused ultrasound (HIFU) treatment that selectively destroys blood clots while minimizing generation of microparticles, or microemboli, that can cause further complications postsurgery. Treatment of malignant blood clots (thrombi) and the resulting emboli are critical problems for numerous patients, and treatments addressing these conditions would benefit from advancements in noninvasive procedures such as HIFU. While recanalization of occlusive blood clots is currently addressed with surgical intervention that seeks to minimize formation of large emboli, there is a danger of microemboli (micrometer-size particles) that have been theorized to be responsible for the poor correlation between apparent surgical success and patient outcome. Here, the addition of phospholipid-coated hydrophobically modified silica nanoparticles (P@hMSNs) improved the efficacy of HIFU treatment by serving as cavitation nuclei for mechanical disruption of thrombi. This treatment was evaluated for the ability to clear the HIFU focal area of a thick and dense thrombus within 10 min. Moreover, it was found that the use of P@hMSN+HIFU treatment generated a significantly smaller microembolic load as compared to comparison techniques, including a HIFU + microbubble contrast agent, HIFU alone, and direct mechanical disruption. This reduction in the microembolic load can occur either with primary removal of the clot by P@hMSN+HIFU or by insonation of the clot fragments after mechanical thrombectomy. Lastly, this method was evaluated in a flow model, where nonocclusive model thrombi and model emboli were mechanically ablated within the focal area within 15 s. Together, these results represent a combination therapy capable of resolving thrombi and microembolisms resulting from thrombectomy through localized destruction of clotted material.
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Embolia/cirurgia , Ablação por Ultrassom Focalizado de Alta Intensidade , Interações Hidrofóbicas e Hidrofílicas , Nanopartículas/química , Fosfolipídeos/química , Dióxido de Silício/química , Trombectomia , Trombose/cirurgia , Animais , Bovinos , Nanopartículas/ultraestruturaRESUMO
Biomimetic colloidal particles are promising agents for biosensing, but current technologies fall far short of Nature's capabilities for sensing, assessing, and responding to stimuli. Phospholipid-containing cell membranes are capable of binding and responding to an enormous variety of biomolecules by virtue of membrane organization and the presence of receptor proteins. By tuning the composition and functionalization of simulated membranes, soft colloids such as droplets and bubbles can be designed to respond to various stimuli. Moreover, because lipid monolayers can surround almost any hydrophobic phase, the interior of the colloid can be selected to provide a sensitive readout, for example in the form of optical microscopy or acoustic detection. In this work, we review some advances made by our group and others in the formulation of lipid-coated particles with different internal phases such as fluorocarbons, hydrocarbons, or liquid crystals. In some cases, binding or displacement of stabilizing lipids gives rise to conformational changes or disruptions in local membrane geometry, which can be amplified by the interior phase. In other cases, multivalent analytes can promote aggregation or even membrane fusion, which can be utilized for optical or acoustic readout. By highlighting a few recent examples, we hope to show that lipid monolayers represent an extremely versatile biosensing platform that can react to and detect biomolecules by leveraging the unique capabilities of phospholipid membranes.
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This review focuses on different materials and contrast agents that sensitize imaging and therapy with Focused Ultrasound (FUS). At high intensities, FUS is capable of selectively ablating tissue with focus on the millimeter scale, presenting an alternative to surgical intervention or management of malignant growth. At low intensities, FUS can be also used for other medical applications such as local delivery of drugs and blood brain barrier opening (BBBO). Contrast agents offer an opportunity to increase selective acoustic absorption or facilitate destructive cavitation processes by converting incident acoustic energy into thermal and mechanical energy. First, we review the history of FUS and its effects on living tissue. Next, we present different colloidal or nanoparticulate approaches to sensitizing FUS, for example using microbubbles, phase-shift emulsions, hollow-shelled nanoparticles, or hydrophobic silica surfaces. Exploring the science behind these interactions, we also discuss ways to make stimulus-responsive, or "turn-on" contrast agents for improved selectivity. Finally, we discuss acoustically-active hydrogels and membranes. This review will be of interest to those working in materials who wish to explore new applications in acoustics and those in acoustics who are seeking new agents to improve the efficacy of their approaches.
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Barreira Hematoencefálica/efeitos da radiação , Sistemas de Liberação de Medicamentos/métodos , Ablação por Ultrassom Focalizado de Alta Intensidade/métodos , Nanopartículas/química , Neoplasias/terapia , Nanomedicina Teranóstica/métodos , Acústica/instrumentação , Animais , Barreira Hematoencefálica/metabolismo , Meios de Contraste/administração & dosagem , Meios de Contraste/química , Humanos , Hidrogéis/administração & dosagem , Hidrogéis/química , Microbolhas , Nanopartículas/administração & dosagem , Neoplasias/metabolismo , Neoplasias/patologia , Permeabilidade/efeitos da radiação , Dióxido de Silício , Nanomedicina Teranóstica/instrumentação , Ondas UltrassônicasRESUMO
In this Letter, we report that surface-bound nanobubbles reduce protein denaturation on methylated glass by irreversible protein shell formation. Single-molecule total internal reflection fluorescence (SM-TIRF) microscopy was combined with intramolecular Förster resonance energy transfer (FRET) to study the conformational dynamics of nitroreductase (NfsB) on nanobubble-laden methylated glass surfaces, using reflection brightfield microscopy to register nanobubble locations with NfsB adsorption. First, NfsB adsorbed irreversibly to nanobubbles with no apparent desorption after 5 h. Moreover, virtually all (96%) of the NfsB molecules that interacted with nanobubbles remained folded, whereas less than 50% of NfsB molecules remained folded in the absence of nanobubbles on unmodified silica or methylated glass surfaces. This trend was confirmed by ensemble-average fluorometer TIRF experiments. We hypothesize that nanobubbles reduce protein damage by passivating strongly denaturing topographical surface defects. Thus, nanobubble stabilization on surfaces may have important implications for antifouling surfaces and improving therapeutic protein storage.
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Nanoestruturas/química , Nitrorredutases/química , Adsorção , Transferência Ressonante de Energia de Fluorescência , Vidro/química , Conformação Proteica , Desnaturação Proteica , Dióxido de Silício/química , Propriedades de SuperfícieRESUMO
In this paper, we report ultrasonically active nanoscale contrast agents that behave as thermometric sensors through phase change in their stabilizing phospholipid monolayer. Phospholipid-stabilized, hydrophobic mesoporous silica nanoparticles (P@hMSNs) are known to interact with high-intensity focused ultrasound (HIFU) to promote cavitation at their surfaces, which can be used for both imaging and therapy. We show that the lateral lipid phase behavior of the phosphocholine lipid dictates the acoustic contrast of the P@hMSNs. When the lipids are in the gel phase below their melting temperature, the P@hMSNs generate detectable microbubbles when exposed to HIFU. However, if the lipids exhibit a liquid expanded phase, the P@hMSNs cease to generate bubbles in response to HIFU insonation. We verify that the heating and subsequent transition of lipid coating the hMSN are associated with the loss of acoustic response by doping laurdan dye into the lipid monolayer and imaging lipid phase through red shifts in emission spectra. Similarly, cessation of cavitation was also induced by adding a fluidizing surfactant such as Triton X, which could be reversed upon washing away the excess surfactant. Finally, by controlling for the partial fluidization caused by the adsorption of protein, P@hMSNs may be used as thermometric sensors of the bulk fluid temperature. These findings not only impact the utilization of nanoscale agents as stimulus-responsive ultrasound contrast agents but also have broader implications for how cavitation may be initiated at surfaces coated by a surfactant.
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The simultaneous delivery of multiple therapeutics to a single site has shown promise for cancer targeting and treatment. However, because of the inherent differences in charge and size between drugs and biomolecules, new approaches are required for colocalization of unlike components in one delivery vehicle. In this work, we demonstrate that triblock copolymers containing click nucleic acids (CNAs) can be used to simultaneously load a prodrug enzyme (cytosine deaminase, CodA) and a chemotherapy drug (doxorubicin, DOX) in a single polymer nanoparticle. CNAs are synthetic analogs of DNA comprised of a thiolene backbone and nucleotide bases that can hybridize to complementary strands of DNA. In this study, CodA was appended with complementary DNA sequences and fluorescent dyes to allow its encapsulation in PEG-CNA-PLGA nanoparticles. The DNA-modified CodA was found to retain its enzyme activity for converting prodrug 5-fluorocytosine (5-FC) to active 5-fluorouracil (5-FU) using a modified fluorescent assay. The DNA-conjugated CodA was then loaded into the PEG-CNA-PLGA nanoparticles and tested for cell cytotoxicity in the presence of the 5-FC prodrug. To study the effect of coloading DOX and CodA within a single nanoparticle, cell toxicity assays were run to compare dually loaded nanoparticles with nanoparticles loaded only with either DOX or CodA. We show that the highest level of cell death occurred when both DOX and CodA were simultaneously entrapped and delivered to cells in the presence of 5-FC.