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A Gold Nanoparticle Bio-Optical Transponder to Dynamically Monitor Intracellular pH
Abstract
A pH-sensitive bio-optical transponder (pH-BOT) capable of simultaneously reporting the timing of intracellular DNA cargo release from a gold nanoparticle (AuNP) and the evolving intracellular pH during endosomal maturation is demonstrated. The pH-BOT is designed with a triple dye-labeled duplex DNA appended to a 6.6 nm AuNP, utilizing pH-responsive fluorescein paired with DyLight405 as a Surface Energy Transfer (SET) coupled dye pair to ratiometrically report the pH at and after cargo release. A non-SET-coupled dye, DyLight700, is used to provide active tracking throughout the experiment. The pH-BOT beacon of the cargo uptake, release, and processing was visualized using live-cell confocal fluorescent microscopy in Chinese hamster ovarian cells, and it was observed that while maturation of endosomes carrying pH-BOT is slowed significantly, the pH-BOT is distributed throughout the endolysosomal system while remaining at pH ~6 i. This observed decoupling of endosomal maturation from acidification lends support to those models which propose that pH alone is not sufficient to explain endosomal maturation and may enable new insights into our understanding of the fundamental processes of biology.
... 1−3 Most NPs enter cells via endocytosis. 4 Once they are internalized, fast acidification of encapsulated NPs via modulation of V-ATPase proton pump activity takes place during the endosome maturation. 5 The process begins from early endosomes (EEs), with a pH value of around 6−6.8, then proceeds to late endosomes (LEs), with a pH of approximately 5.2−6, and finally to, the lysosome (Lys), capable of acidification to a pH of 4.5−5.2. ...
... Several control experiments were performed before implementing triple-labeled MSNs in intracellular studies. 4,11,13,15 Different excitation laser powers, from 0.4 to 0.8 μW, were used to excite the triple-labeled MSN@PEI. The results showed a stable intensity ratio in these ranges ( Figure S5a), which suggests that fluctuations of the excitation laser's power did not alter the pH readouts. ...
... Moreover, immunostaining of EEA-1 in EE and LAMP-1 in the lysosome membrane was also performed to visualize the intracellular distribution (Figure 9b). 3,4 Less than 7% of the colocalization of MSN@PEI@L with EEA-1 showed their capability to escape from early endosome and stay diffused in the entire region of the cell, which probably indicates the cytosol. On the other hand, MSN@PEI@L (34.2%) were found to be more co-localized with LAMP-1 compared to the other two MSNs, which supports lysosomal sorting peptides (YQRLGC peptides) playing an important role in targeting the lysosome, as illustrated in Figure 9c. ...
Nanoparticles (NPs)-based targeted drug delivery is intended to transport therapeutically active molecules to specific cells, and particular intracellular compartments. However, there is limited knowledge regarding the complete route of NPs in this targeting scenario. In this study, simultaneously performing motion and dynamic pH sensing using single-particle tracking (SPT) leads to an alternative method of gaining insight on the mesoporous silica nanoparticle’s (MSN) journey in targeting lysosome. Two different pH-sensitive dyes and a reference dye, are incorporated into mesoporous silica nanoparticles (MSNs) via co-condensation to broaden the measurable pH range (pH 4~7.5) of nanoprobe. The phosphonate, amine, and lysosomal sorting peptides (YQRLGC) are conjugated onto MSN’s surface to study intracellular nano-bio interactions of two oppositely charged- and lysosome targetable-MSN, respectively. The brightness and stability of these MSNs allow their movement and dynamic pH evolution during their journey to be simultaneously monitored in real-time. Importantly, a multidimensional analysis of MSN's movement and local-pH have revealed a new model intracellular dynamic states and distributions of MSNs, previously inaccessible when using single parameters alone. A key result is that YQRLGC-conjugated MSNs took an alternative route to target lysosomes apart from the traditional one, which sped up to 4h and enhanced their targeting efficiency (up to 32%). The findings enrich our understanding of the intracellular journey of MSNs. This study offers complementary information on correlating the surface design with the full pathway of nanoparticles to achieve targeted delivery of therapeutic payload.
... However, endocytosis of probes can be challenging, and intracellular growth of the nanoparticle may be an interesting alternative (20). Once internalized, functional AuNPs are an excellent strategy to report on various molecular targets within cells, such as pH (21)(22)(23). While possibilities of using SERS to report on intracellular chemicals are essentially limitless, there are challenges with the internalization process and with spatially addressing the response from the SERS substrate, which are barriers in some applications of SERS for biochemical or clinical diagnostics. ...
A frontier of analytical sciences is centered on the continuous measurement of molecules in or near cells, tissues, or organs, within the biological context in situ, where the molecular-level information is indicative of health status, therapeutic efficacy, and fundamental biochemical function of the host. Following the completion of the Human Genome Project, current research aims to link genes to functions of an organism and investigate how the environment modulates functional properties of organisms. New analytical methods have been developed to detect chemical changes with high spatial and temporal resolution, including minimally invasive surface-enhanced Raman scattering (SERS) nanofibers using the principles of endoscopy (SERS nanoendoscopy) or optical physiology (SERS optophysiology). Given the large spectral data sets generated from these experiments, SERS nanoendoscopy and optophysiology benefit from advances in data science and machine learning to extract chemical information from complex vibrational spectra measured by SERS. This review highlights new opportunities for intracellular, extracellular, and in vivo chemical measurements arising from the combination of SERS nanosensing and machine learning.
... Gold nanostar (GNS) is attached with Rhodamine 6G (Rh-6G) dye conjugated DNA aptamer. This is because GNS can effectively quench fluorescence signal from the dye via nanoparticle surface energy transfer (NSET) (Carnevale et al., 2018). Based on Figure 18, the aptamer-attached GNS exhibits high Figure 16. ...
Due to the recent COVID-19 pandemic that occurred worldwide since 2020, scientists and researchers have been studying methods to detect the presence of the virus causing COVID-19 disease, namely SARS-CoV-2. Optical spectroscopy is a method that employs the interaction of light in detecting virus on samples. It is a promising method that might help in detecting the presence of SARS-CoV-2 in samples. Four optical spectroscopy methods are discussed in this paper: ultraviolet (UV), infrared (IR), Raman spectroscopy and fluorescence spectroscopy. UV and IR spectroscopy differ in wavelength range (less than 400 nm for UV, more than 700 nm for IR). Raman spectroscopy involves shift in wavelength due to scattering of light. Fluorescence spectroscopy involves difference in wavelength between absorbed and emitted light due to vibrational relaxation. These four methods had been proven to differentiate healthy samples from virus-infected samples. UV spectroscopy is useful in determining presence of virus based on 260 nm/280 nm absorbance ratio. However, its usefulness is limited due to its destructive properties on virus at sufficiently high intensity. Meanwhile, IR spectroscopy has becoming popular in studies involving virus samples. Mid-infrared (MIR) spectroscopy is most commonly used among IR spectroscopy as it usually provides useful information directly from spectral data. Near infrared (NIR) spectroscopy is also used in studying virus samples, but additional methods such as principal component analysis (PCA) and partial least squares (PLS) are required to process raw spectral data and to identify molecules based on spectral peaks. On the other hand, Raman spectroscopy is useful because spectral data can be analyzed directly in identifying vibrational modes of specific molecules in virus samples. Fluorescence spectroscopy relies on interaction between viral particles and fluorescent tags for the detection of virus based on improvement or quenching of fluorescent signal. Due to non-invasive properties of virus samples, IR, Raman and fluorescence spectroscopy will be used more often in future studies involving virus detection in infected samples.
... Gold nanostar (GNS) is attached with Rhodamine 6G (Rh-6G) dye conjugated DNA aptamer. This is because GNS can effectively quench fluorescence signal from the dye via nanoparticle surface energy transfer (NSET) (Carnevale et al., 2018). Based on Figure 18, the aptamer-attached GNS exhibits high Figure 16. ...
... 17 For example, numerous nanoparticle-based nanomaterials have been used to fabricate nanoprobes capable of monitoring pH uctuations during important physiological processes such as the cell cycle or cellular autophagy, 10,18 as well as tracking the maturation process of endosomes. 19,20 Despite the great progress, the development of nanoprobes for real-time tracking of pH changes still suffers from several limitations. The rst limitation is the stability of the probes; the commonly used probes based on uorescent dyes or uorescent proteins 21,22 are still not immune to the effects of photobleaching and cellular autouorescence, 23 and some organic small molecule dyes have a tendency to rapid leak from cells. ...
Understanding the pH evolution during endocytosis is essential for our comprehension of the fundamental processes of biology as well as effective nanotherapeutic design. Herein, we constructed a plasmonic Au@PANI core-shell nanoprobe, which possessed significantly different scattering properties under acidic and basic conditions. Encouragingly, the scattering signal of Au@PANI nanoprobes displayed a positive linear correlation with the pH value not only in PBS but also in nigericin-treated cells. Ultimately, benefiting from the excellent response properties as well as the excellent biocompatibility and stability, the Au@PANI nanoprobes have successfully enabled a dynamic assessment of the evolving pH in the endosomal package as the endosome matured from early to late, and eventually to the lysosome, by reporting scattering signal changes.
... Based on the unique optical properties of AuNPs, many colorimetric methods have been established for assaying the different kinds of species like metal ions, proteins, nucleic acids, and cells [5][6][7]. In addition, the fluorescence resonance energy transfer (FRET) quenching mechanisms of AuNPs makes it useful in the bioimaging system for assaying pH, proteins and DNA [8][9][10]. Since AuNPs are inert and biocompatible, the aggregation of AuNPs can form highly efficient near-infrared (NIR) photothermal transducers for photothermal therapy [11][12][13]. ...
Background:
Gold nanoparticles (AuNPs) have been proposed for many applications in medicine and bioanalysis. For use in all these applications, maintaining the stability of AuNPs in solution by suppressing aggregation is paramount. Herein, the effects of amino acids were investigated in stabilizing AuNPs by rationally designed peptide scaffolds.
Results:
Compared to other tested amino acids, phosphotyrosine (pY) significantly stabilized AuNPs. Our results indicated that pY modified AuNPs presented a high level of stability in various solutions, and had good biocompatibility. When a pY-peptide was used in stabilizing AuNPs, the phosphate group could be removed by phosphatases, which subsequently caused the aggregation and the cargo release of AuNPs. In vitro study showed that AuNPs formed aggregation in a phosphatase concentration depending manner. The aggregation of AuNPs was well correlated with the enzymatic activity (R2 = 0.994). In many types of cancer, a significant increase in phosphatases has been observed. Herein, we demonstrated that cancer cells treated with pY modified AuNPs in conjunction with doxorubicin killed SGC-7901 cells with high efficiency, indicating that the pY peptide stabilized AuNPs could be used as carriers for targeted drug delivery.
Conclusion:
In summary, pY peptides can act to stabilize AuNPs in various solutions. In addition, the aggregation of pY-AuNPs could be tuned by phosphatase. These results provide a basis for pY-AuNPs acting as potential drug carriers and anticancer efficacy.
... In addition, this protocol for unspecific labelling of outer cell membrane proteins can be adapted for the use of different types of gold nanosensor, which can vary in size, morphology and choice of SERS active compound. Recently, Carnevale et al. 56 developed an advanced method for intracellular pH measurement based on surface energy transfer using AuNP and fluorescence dyes, in which the rates of laser-induced photo-bleaching were reduced during multihour live-cell experiments. This method may certainly be extended to the measurement of cell surface pH by taking advantage of part of the knowledge introduced in our paper. ...
Regulation of intracellular pH is critically important for many cellular functions. The quantification of proton extrusion in different types of cells and physiological conditions is pivotal to fully elucidate the mechanisms of pH homeostasis. Here we show the use of gold nanoparticles (AuNP) to create a high spatial resolution sensor for measuring extracellular pH in proximity of the cell membrane. We tested the sensor in HepG2 liver cancer cells and MKN28 gastric cancer cells before and after inhibition of Na+/H+ exchanger. The gold surface conjugation strategy is conceived with a twofold purpose: i) to attach the AuNP to the membrane proteins and ii) to quantify the local pH from AuNP using surface enhanced Raman spectroscopy (SERS). The nanometer size of the cell membrane anchored sensor and the use of SERS enable us to visualize highly localized variation of pH induced by H+ extrusion, which is particularly upregulated in cancer cells.
In the biological system, the intracellular pH (pHi) plays an important role in regulating diverse physiological activities, including enzymatic action, ion transport, cell proliferation, metabolism, and programmed cell death. The monitoring of pH inside living cells is also crucial for studying cellular events such as phagocytosis, endocytosis, and receptor-ligand internalization. Furthermore, some organelles, viz., endosomes and lysosomes, have intracompartmental pH, which is critical for maintaining the stability of protein structure and function. The dysfunction and abnormal pH regulation can result in terminal diseases such as cancer, Alzheimer, and so forth. Therefore, the accuracy of intracellular pH measurement is always the top priority and demands cutting-edge research and analysis. Such techniques, such as Raman spectroscopy and fluorescence imaging, preferably use nanotechnology due to their remarkable advantages, such as a non-invasive approach and providing accuracy, repeatability, and reproducibility. In the past decades, there have been numerous attempts to design and construct non-invasive organic and inorganic materials-based nanoprobes for pHi sensing. For Raman-based techniques, metal nanostructures such as Au/Ag/Cu nanoparticles are utilized to enhance the signal intensity. As for the fluorescence-based studies, the organic-based small molecules, such as dyes, show higher sensitivity toward pH. However, they possess several drawbacks, including high photobleaching rate, and autofluorescence background signals. To this end, there are alternative nanomaterials proposed, including semiconductor quantum dots (QDs), carbon QDs, upconversion nanoparticles, and so forth. Moreover, the fluorescence technique allows for ratiometric measurement of pHi, which as a result, offers a reliable calibration curve. This timely review will critically examine the current progression in the existing nanoprobes. In addition, based on our knowledge and available research findings, we provide a brief future outlook that may advance the state-of-the-art methodologies for pHi sensing.
RNA interference (RNAi) has increased the possibility of restoring RNA drug targets for cancer treatment. Small interfering RNA (siRNA) is a promising therapeutic RNAi tool that targets the defective gene by inhibiting its mRNA expression and stopping its translation. However, siRNAs have flaws like poor intracellular trafficking, RNase degradation, rapid kidney filtration, off-targeting, and toxicity, which limit their therapeutic efficiency. Nanocarriers (NCs) have been designed to overcome such flaws and increase antitumor activity. Combining siRNA and anticancer drugs can give synergistic effects in cancer cells, making them a significant gene-modification tool in cancer therapy. Our discussion of NCs-mediated siRNA delivery in this review includes their mechanism, limitations, and advantages in comparison with naked siRNA delivery. We will also discuss organic NCs (polymers and lipids) and inorganic NCs (quantum dots, carbon nanotubes, and gold) that have been reported for extensive delivery of therapeutic siRNA to tumor sites. Finally, we will conclude by discussing the studies based on organic and inorganic NCs-mediated siRNA drug delivery systems conducted in the years 2020 and 2021.
There is significant interest in developing photothermal systems that can precisely control the structure and function of biomolecules through local temperature modulation. One specific application is the denaturation of double-stranded (ds) DNA through femtosecond (fs) laser pulse optical heating of gold nanoparticles (AuNPs); however, the mechanism of DNA melting in these systems is not fully understood. Here, we utilize 55 nm AuNPs with surface-tethered dsDNA, which are locally heated using fs laser pulses to induce DNA melting. By varying the dsDNA distance from the AuNP surface and the laser pulse energy fluence, this system is used to study how the nanosecond duration temperature increase and the steep temperature gradient around the AuNP affect dsDNA dehybridization. Through modifying the distance between the dsDNA and AuNP surface by 3.8 nm in total and the pulse energy fluence from 7.1 to 14.1 J/m2, the dehybridization rates ranged from 0.002 to 0.05 DNA per pulse, and the total amount of DNA released into solution was controlled over a range of 26-93% in only 100 s of irradiation. By shifting the dsDNA position as little as ∼1.1 nm, the average dsDNA dehybridization rate is altered up to 30 ± 2%, providing a high level of control over DNA melting and release. By comparing the theoretical temperature around the dsDNA to the experimentally derived temperature, we find that maximum or peak temperatures have a greater influence on the dehybridization rate when the dsDNA is closer to the AuNP surface and when lower laser pulse fluences are used. Furthermore, molecular dynamics simulations mimicking the photothermal heat pulse around a AuNP provide mechanistic insight into the stochastic nature of dehybridization and demonstrate increased base pair separation near the AuNP surface during laser pulse heating when compared to steady-state heating. Understanding how biological materials respond to the short-lived and non-uniform temperature increases innate to fs laser pulse optical heating of AuNPs is critical to improving the functionality and precision of this technique so that it may be implemented into more complex biological systems.
Gold nanoparticles (AuNPs) pose a great challenge in the development of nanotracers that can self‐adaptively alter their properties in response to certain cellular environments for long‐term stem cell tracking. Herein, pH‐sensitive Au nanotracers (CPP‐PSD@Au) are fabricated by sequential coupling of AuNPs with sulfonamide‐based polymer (PSD) and cell‐penetrating peptide (CPP), which can be efficiently internalized by mesenchymal stem cells (MSCs) and undergo pH‐induced self‐assembly in endosomes, facilitating long‐term computed tomography (CT) imaging tracking MSCs in a murine model of idiopathic pulmonary fibrosis (IPF). Using the CPP‐PSD@Au, the transplanted MSCs for the first time can be monitored with CT imaging for up to 35 days after transplantation into the lung of IPF mice, clearly elucidating the migration process of MSCs in vivo. Moreover, we preliminarily explored the mechanism of the CPP‐PSD@Au labeled MSCs in the alleviation of IPF, including recovery of alveolar integrity, decrease of collagen deposition, as well as down‐regulation of relevant cytokine level. This work facilitates our understanding of the behavior and effect of MSCs in the therapy of IPF, thereby providing an important insight into the stem cell‐based treatment of lung diseases.
We herein innovatively constructed the pure pH-dual channel probe DNOXA by the reaction of oxalyl chloride with 4-dimethylaminobenzaldehyde. DNOXA has two response sites for acidity, which are corresponding to pH-dual channel responses. When adding acid to pH 5.0, the protonation in the response site I enhanced the push-pull effect, and the absorption maxima of the optical spectra bathochromically shifted from 463 to 652 nm. When sequentially adding acid to pH 1.6, the response site II is awakened to work. The enhanced push-pull system was interrupted, and the absorption maxima hypochromatically shifted from 652 to 506 nm. In this process, the ratiometric change in the probe can be observed by naked-eye. Furthermore, quantum-chemical study well explained and supported the difference of their maxima absorption. Based on the excellent response of DNOXA to pH in vitro, DNOXA had commendable specific imaging and localization ability for lysosomes, undergoing dual channel images of protonation at the acidic microenvironment.
The ongoing outbreak of the coronavirus infection has killed more than 2 million people. Herein, we demonstrate that Rhodamine 6G (Rh-6G) dye conjugated DNA aptamer-attached gold nanostar (GNS)-based distance-dependent nanoparticle surface energy transfer (NSET) spectroscopy has the capability of rapid diagnosis of specific SARS-CoV-2 spike recombinant antigen or SARS-CoV-2 spike protein pseudotyped baculovirus within 10 min. Because Rh-6G-attached single-stand DNA aptamer wrapped the GNS, 99% dye fluorescence was quenched because of the NSET process. In the presence of spike antigen or virus, the fluorescence signal persists because of the aptamer-spike protein binding. Specifically, the limit of detection for the NSET assay has been determined to be 130 fg/mL for antigen and 8 particles/mL for virus. Finally, we have demonstrated that DNA aptamer-attached GNSs can stop virus infection by blocking the angiotensin-converting enzyme 2 (ACE2) receptor binding capability and destroying the lipid membrane of the virus.
A comprehensive understanding of interactions between nanoparticles (NPs) and biological components is critical to the clinical application of NPs and nanomedicine. Here we provide a step-by-step correlative imaging approach to investigate plasmonic NPs of different aggregation states at the single-cell level. Traceable spherical nucleic acids (SNAs) are fabricated by decorating 50-nm spherical gold NPs with fluorophore-labeled DNA, serving as dually emissive (fluorescent and plasmonic) NPs. The in situ correlative imaging with dark-field microscopy (DFM) and fluorescence microscopy (FM) reveals intracellular distribution of SNAs, whereas DFM combined with scanning electron microscopy (SEM) allows semi-quantification of SNA clustering states in solution. The imaging data are analyzed by ImageJ and a colorimetry-based algorithm written in Python. The clustering states of SNAs in a single cell can be efficiently distinguished within 20 s. This method can be readily installed to monitor real-time endocytosis and cellular distribution of plasmonic NPs of different aggregation states and to quantitatively image targets of interest (e.g., specific DNA, messenger RNA, peptides or proteins) in living cells. The entire procedure can be completed in 3–5 d and requires standard DFM, FM and SEM imaging and data analysis skills and equipment.
This is the first comprehensive review on methods and materials for use in optical sensing of pH values, and on applications of such sensors. The Review starts with an introduction that contains subsections on the definition of the pH value, a brief look back on optical methods for sensing of pH, on the effects of ionic strength on pH values and pKa values, on the selectivity, sensitivity, precision, dynamic ranges and temperature dependence of such sensors. Commonly used optical sensing schemes are covered in a next main chapter, with subsections on methods based on absorptiometry, reflectometry, luminescence, refractive index, surface plasmon resonance, photonic crystals, turbidity, mechanical displacement, interferometry and solvatochromism. This is followed by sections on absorptiometric and luminescent molecular probes for use pH in sensors. Further large sections cover polymeric hosts and supports, and methods for immobilization of indicator dyes. Further and more specific sections summarize the state of the art in materials with dual functionality (indicator and host), nanomaterials, sensors based on upconversion and 2-photon absorption, multiparameter sensors, imaging, and sensors for extreme pH values. A chapter on the many sensing formats has subsections on planar, fiber optic, evanescent wave, refractive index, surface plasmon resonance and holography based sensor designs, and on distributed sensing. Another section summarizes selected applications in areas such as medicine, biology, oceanography, bioprocess monitoring, corrosion studies, on the use of pH sensors as transducers in biosensors and chemical sensors, and their integration into flow-injection analyzers, microfluidic devices and lab-on-a-chip systems. An extra section is devoted to current challenges, with subsections on challenges of general nature and those of specific nature. A concluding chapter gives an outlook on potential future trends and perspectives.
Visualization of intracellular pH (i-pH) using surface-enhanced Raman spectroscopy (SERS) plays an important role toward understanding of cellular processes including their interactions with nanoparticles. However, conventional two-dimensional SERS imaging often fails to take into consideration changes occurring in the whole-cell volume. We therefore aimed at obtaining a comprehensive i-pH profile of living cells by means of three-dimensional (3D) SERS imaging, thereby visualizing dynamic i-pH distribution changes in a single cell. We devised here a biocompatible and highly stable SERS pH probe, comprising plasmonic gold nanostars functionalized with a pH-sensitive Raman reporter tag-4-mercaptobenzoic acid-and protected by a cationic biocompatible polymer, poly-l-arginine hydrochloride (PA). The positively charged PA coating plays a double role in enhancing cell uptake and providing chemical and colloidal stability in cellular environments. The SERS-active pH probe allowed visualization of local changes in i-pH, such as acidification during nanoparticle (NP) endocytosis. We provide evidence of i-pH changes during NP endocytosis via high-resolution 3D SERS imaging, thereby opening new avenues toward the application of SERS to intracellular studies.
Understanding the initial adsorption and subsequent translocation of nanoparticles (NPs) across the phospholipid bilayer of liposome find importance in drug delivery and in vivo bio-imaging applications. Herein, we have investigated the time-dependent selective uptake of 2.0 ± 0.3 nm-sized CdTe quantum dots (QDs) into small unilamellar vesicles (SUV) of dipalmitoylphoshatidylcholine (DPPC) in its gel phase and subsequent alteration of nanometal surface energy transfer (NSET) in the presence of 18.0 ± 0.2 nm-sized citrate-capped gold NPs (Au NPs). The interactions between CdTe QD and Au NP across the lipid bilayer of DPPC liposome has been explored by monitoring the NSET process using fluorescence spectroscopy. The time-dependent selective partitioning of QDs into the liposomal phase has been demonstrated using confocal laser scanning microscopy (CLSM) and atomic force microscopy (AFM). It has been observed that instant mixing of QDs and Au NPs with liposomes does not alter the extent of NSET between QD-Au NP pair relative to that in the absence of liposome. However, equilibrating the binary mixture of liposome and QDs for 24 h at room temperature leads to the formation of liposome encapsulated QD (LipQD) which does not take part in NSET with aqueous phase Au NPs. In contrast, equilibrating the ternary mixture of QDs, liposomes, and Au NPs for 24 h results in the formation of Au NP adsorbed QD-encapsulated liposomes (AuLipQD) which exhibit moderate amount of NSET from encapsulated QDs to the adsorbed Au NPs at the surface of liposomes. It has been observed that the efficiency of NSET decreases from 62% in bulk aqueous medium to 24% in AuLipQD complex. Our present findings may be useful to understand the fundamental interaction of other metal and semiconductor NPs with liposomes and cell membrane for various drug-delivery and bio-imaging applications.
Femtosecond (fs) laser pulsed excitation of plasmonic nanoparticle (NP)-biomolecule conjugates is a promising method to locally heat biological materials. Studies have demonstrated that fs pulses of light can modulate the activity of DNA or proteins when attached to plasmonic NPs; however, the precision over subsequent biological function remains largely undetermined. Specifically, the temperature the localized biomolecules "experience" remains unknown. We used 55 nm gold nanoparticles (AuNPs) displaying double-stranded (ds) DNA to examine how, for dsDNA with different melting temperatures, the laser pulse energy fluence and bulk solution temperature affect the rate of local DNA denaturation. A universal "template" single-stranded DNA was attached to the AuNP surface, and three dye-labeled probe strands, distinct in length and melting temperature, were hybridized to it creating three individual dsDNA-AuNP bioconjugates. The dye-labeled probe strands were used to quantify the rate and amount of DNA release after a given number of light pulses, which was then correlated to the dsDNA denaturation temperature, resulting in a quantitative nanothermometer. The localized DNA denaturation rate could be modulated by more than threefold over the biologically relevant range of 8-53 °C by varying pulse energy fluence, DNA melting temperature, and surrounding bath temperature. With a modified dissociation equation tailored for this system, a "sensed" temperature parameter was extracted and compared to simulated AuNP temperature profiles. Determining actual biological responses in such systems can allow researchers to design precision nanoscale photothermal heating sources.
Due to the remarkable properties of gold nanoparticles (AuNPs), they have been extensively employed as a potential tool for the diagnosis and treatment of cancers. While the conventional drug delivery system fails to efficiently deliver the chemotherapeutics due to the complexity of tumor microenvironment, people have been seeking alternative nanoparticulate strategy to improve the tumor specificity, the therapeutic index as wells as the pharmacokinetic profile. The nontoxic and nonimmunogenic nature, the high permeability, and superior retention effect of AuNPs provide additional benefits by enabling easy penetration and accumulation of drugs at the tumor sites. Here in this review, we focused on discussing the recent advances regarding the design of acid‐responsive AuNPs as well as their applications in drug/gene delivery and imaging in cancer diagnosis and therapy. Particularly, we highlighted the size switch strategies used in the development of acid‐responsive AuNPs.
This article is categorized under:
• Diagnostic Tools > in vivo Nanodiagnostics and Imaging
• Therapeutic Approaches and Drug Discovery > Nanomedicine for Oncologic Disease
There is a large number of two‐dimensional static in vitro studies about the uptake of colloidal nano‐ and microparticles, which has been published in the last decade. In this Minireview, different methods used for such studies are summarized and critically discussed. Supplementary experimental data allow for a direct comparison of the different techniques. Emphasis is given on how quantitative parameters can be extracted from studies in which different experimental techniques have been used, with the goal of allowing better comparison.
There is a large number of 2‐dimensional static in vitro studies about the uptake of colloidal nano‐ and microparticles, which has been published in the last decade. In this viewpoints article different methods used for such studies are summarized and critically discussed. Supplementary experimental data allow for a direct comparison of the different techniques. Emphasis is given on how quantitative parameters can be extracted from studies in which different experimental techniques have been used, with the goal of allowing better comparison.
The accurate detection of intracellular and extracellular microenvironment at single cell is important for deep understanding of complicated cellular physiological activities, such as exploring organelle structures, and revealing the spatial heterogeneity of intracellular chemical components. Here, we built a single cell analysis platform by using a silver nanoparticles decorated optical fiber tip (diameter less than 500 nm) integrated with microoperation, microspectroscopy and microimaging, working for light propagation, sensing, cell manipulation, and in-situ spectroscopy and imaging, especially for exploring the subcellular microenvironment of a single cell. In view of the flexibility of fiber tip, the silver nanoparticles and the pH sensitive Raman reporters were both modified on the fiber tip surface by the one-step laser-induced Ag deposition method, and this optical fiber sensor was used for the accurate intracellular and extracellular pH detections by the surface-enhanced Raman spectroscopy (SERS) method. Cell heterogeneity according to their different pH microenvironments was disclosed by this optical fiber-based single cell analysis platform and it is also promising in learning biological processes within single cell, as well as monitoring intracellular responses during various external stimuli and therapies.
Covalently coupling nucleic acids to a gold nanoparticle (AuNP) surface has been demonstrated as an effective gene therapy agent to modify cellular protein expression. The therapeutic efficacy of the approach is anticipated to be impacted by the length of time the nucleic acid sequence resides in the endo-lysosomal pathway once transfected into a cell. It is believed that the dynamics of the processing should reflect the linkage chemistry of the DNA to the AuNP surface. In this manuscript the dynamics of nanotherapeutic uptake, nucleic acid release, and gene processing are investigated in vitro for a AuNP-nucleic acid delivery platform transfected into A375 human melanoma cells, as a function of the nucleic acid-gold linkage chemistry. The dynamics of cell processing of the single monodentate thiol (SX), bidentate dual thiol (SS), or mixed bidentate thiol plus amine (SN) coordination of nucleic acids to the AuNP surface are evaluated using a multicolor nano-surface energy transfer bio-optical transponder (SET-BOT) technology. The use of live-cell fluorescence microscopy allows for the direct visualization of the uptake and localization of a lipofectamine packaged SET-BOT using a dye (DL700) that is not quenched in the proximity of the AuNP, while fluorescence “turn on” of a dye that is proximally quenched by the AuNP (DL488) is used to report on the dynamics of release of the nucleic acid cargo within the cell. For protein expression following transcription of the gene, the emission signature of a red fluorescent protein, tdTomato, is monitored. The intracellular rates of DNA release from the AuNP surface once endosomally packaged within the A375 human melanoma cells were found to follow the binding activity series: bidentate thiol > bidentate thiol plus amine > monodentate thiol, consistent with the strength of multidentate chelation, paired with the stabilizing influence of pi-backbonding of thiols compared to sigma donation in amines, when bound to a gold surface.
Single molecule detection methods, such as nanopore sensors have found increasing importance in applications ranging from gaining a better understanding of biophysical processes to technology driven solutions such as DNA sequencing. However, challenges remain especially in relation to improving selectivity to probe specific targets or to alternatively enable detection of smaller molecules such as small-sized proteins with a sufficiently high signal-to-noise ratio. In this article, we propose a solution to these technological challenges by using DNA aptamer-modified gold nanoparticles (AuNPs) that act as a molecular carrier through the nanopore sensor. We show that this approach offers numerous advantages including: high levels of selectivity, efficient capture from a complex mixture, enhanced signal, minimized analyte-sensor surface interactions, and finally can be used to enhance the event detection rate. This is demonstrated by incorporating a lysozyme binding aptamer to a 5 nm AuNP carrier to selectively probe lysozyme within a cocktail of proteins. We show that nanopores can reveal sub-complex molecular information, by discriminating the AuNP from the protein analyte, indicating the potential use of this technology for single molecule analysis of different molecular analytes specifically bound to AuNP.
The design and use of materials in the nanoscale size range for addressing medical and health-related issues continues to receive increasing interest. Research in nanomedicine spans a multitude of areas, including drug delivery, vaccine development, diagnosis and imaging tools, wearable devices, high-throughput screening platforms, etc. using biological, nonbiological, or hybrid materials. Many of these developments are starting to be translated into viable clinical products. Here, we provide an overview of recent developments in nanomedicine and highlight the current challenges and upcoming opportunities for the field and translation to the clinic.
Background
ImageJ is an image analysis program extensively used in the biological sciences and beyond. Due to its ease of use, recordable macro language, and extensible plug-in architecture, ImageJ enjoys contributions from non-programmers, amateur programmers, and professional developers alike. Enabling such a diversity of contributors has resulted in a large community that spans the biological and physical sciences. However, a rapidly growing user base, diverging plugin suites, and technical limitations have revealed a clear need for a concerted software engineering effort to support emerging imaging paradigms, to ensure the software’s ability to handle the requirements of modern science.
Results
We rewrote the entire ImageJ codebase, engineering a redesigned plugin mechanism intended to facilitate extensibility at every level, with the goal of creating a more powerful tool that continues to serve the existing community while addressing a wider range of scientific requirements. This next-generation ImageJ, called “ImageJ2” in places where the distinction matters, provides a host of new functionality. It separates concerns, fully decoupling the data model from the user interface. It emphasizes integration with external applications to maximize interoperability. Its robust new plugin framework allows everything from image formats, to scripting languages, to visualization to be extended by the community. The redesigned data model supports arbitrarily large, N-dimensional datasets, which are increasingly common in modern image acquisition. Despite the scope of these changes, backwards compatibility is maintained such that this new functionality can be seamlessly integrated with the classic ImageJ interface, allowing users and developers to migrate to these new methods at their own pace.
Conclusions
Scientific imaging benefits from open-source programs that advance new method development and deployment to a diverse audience. ImageJ has continuously evolved with this idea in mind; however, new and emerging scientific requirements have posed corresponding challenges for ImageJ’s development. The described improvements provide a framework engineered for flexibility, intended to support these requirements as well as accommodate future needs. Future efforts will focus on implementing new algorithms in this framework and expanding collaborations with other popular scientific software suites.
Electronic supplementary material
The online version of this article (doi:10.1186/s12859-017-1934-z) contains supplementary material, which is available to authorized users.
The ability of Au and other metal nanostructures to strongly quench the fluorescence of proximal fluorophores (dyes and fluorescent proteins) has made AuNP-conjugates attractive for use as platforms for sensor development based on energy transfer interactions. In this study, we first characterize the energy transfer quenching of mCherry fluorescent proteins immobilized on AuNPs via metal-imidazole coordination, where parameters such as NP size and number of attached proteins are varied. Using steady-state and time-resolved fluorescence measurements, we recorded very high mCherry quenching, with efficiency reaching ≈ 95-97%, independent of the NP size or number of bound fluorophores (i.e., conjugate valence). We further exploited these findings to develop a solution phase sensing platform targeting thiolate compounds. Energy Transfer (ET) was employed as a transduction mechanism to monitor the competitive displacement of mCherry from the Au surface upon the introduction of varying amounts of thiolates with different size and coordination numbers. Our results show that the competitive displacement of mCherry depends on the thiolate concentration, time of reaction and type of thiol derivatives used. Further analysis of the PL recovery data provides a measure for the equilibrium dissociation constant (Kd^-1) for these compounds. These findings combined indicate that the AuNP-FP conjugates may offer a potentially useful platform for thiol sensing both in solution and in cell cultures.
Accurate quantitation of intracellular pH (pHi) is of great importance in revealing the cellular activities and early warning of diseases. A series of fluorescence-based nano-bioprobes composed of different nanoparticles or/and dye pairs have already been developed for pHi sensing. Till now, biological auto-fluorescence background upon UV-Vis excitation and severe photo-bleaching of dyes are the two main factors impeding the accurate quantitative detection of pHi. Herein, we have developed a self-ratiometric luminescence nanoprobe based on förster resonant energy transfer (FRET) for probing pHi, in which pH-sensitive fluorescein isothiocyanate (FITC) and upconversion nanoparticles (UCNPs) were served as energy acceptor and donor, respectively. Under 980 nm excitation, upconversion emission bands at 475 nm and 645 nm of NaYF4:Yb3+, Tm3+ UCNPs were used as pHi response and self-ratiometric reference signal, respectively. This direct quantitative sensing approach has circumvented the traditional software-based subsequent processing of images which may lead to relatively large uncertainty of the results. Due to efficient FRET and fluorescence background free, a highly-sensitive and accurate sensing has been achieved, featured by 3.56 per unit change in pHi value 3.0–7.0 with deviation less than 0.43. This approach shall facilitate the researches in pHi related areas and development of the intracellular drug delivery systems.
New achievements in the realm of nanoscience and innovative techniques of nanomedicine have moved micro/nanoparticles (MNPs) to the point of becoming actually useful for practical applications in the near future. Various differences between the extracellular and intracellular environments of cancerous and
normal cells and the particular characteristics of tumors such as physicochemical properties,
neovasculature, elasticity, surface electrical charge, and pH have motivated the design and fabrication of inventive ‘‘smart’’ MNPs for stimulus-responsive controlled drug release. These novel MNPs can be tailored to be responsive to pH variations, redox potential, enzymatic activation, thermal gradients, magnetic fields, light, and ultrasound (US), or can even be responsive to dual or multi-combinations of different stimuli. This unparalleled capability has increased their importance as site-specific controlled drug delivery systems (DDSs) and has encouraged their rapid development in recent years. An in-depth understanding of the underlying mechanisms of these DDS approaches is expected to further contribute to this groundbreaking field of nanomedicine. Smart nanocarriers in the form of MNPs that can be triggered by internal or external stimulus are summarized and discussed in the present review, including pH-sensitive peptides and polymers, redox-responsive micelles and nanogels, thermo- or magnetic-responsive nanoparticles (NPs), mechanical- or electrical-responsive MNPs, light or ultrasound-sensitive particles, and multi responsive
MNPs including dual stimuli-sensitive nanosheets of graphene. This review highlights the recent advances of smart MNPs categorized according to their activation stimulus (physical, chemical, or biological) and looks forward to future pharmaceutical applications.
Fingerprinting biochemical changes associated with cellular responses to external stimuli can provide vital information on the dynamics of biological processes and their defense mechanisms. In this study, surface-enhanced Raman spectroscopy (SERS) has been used to elucidate biomolecular dynamics on the response of healthy and cancerous cells towards ultraviolet (UV) light irradiation at the cellular level in real-time. We have identified a number of physiochemical damages to proteins, especially to the chemical structure of the sulfur and aromatic amino acid containing moieties, as well as changes in secondary structure. Furthermore, we found that continuous exposure of short wave UV-C light (254 nm) to living cells can photolytically damage intracellular proteins and can completely arrest nanoparticle transport and trigger apoptosis. However, under similar conditions, this was not observed when the cells were exposed to long wave UV-A light (365 nm). These biomolecular events were probed in real-time using SERS and dark-field (DF) imaging. Specifically, this technique has been utilized for the real-time evaluation of a unique cellular defense mechanism in cancer cells towards UV exposure. Our technique provides a powerful approach to understand the mechanisms of UV light-triggered cell death, protein dynamics, and enhanced cell repair and defense machinery within cancer cells through actively monitoring molecular vibrations.
The endosomal-lysosomal system is made up of a set of intracellular membranous compartments that dynamically interconvert, which is comprised of early endosomes, recycling endosomes, late endosomes, and the lysosome. In addition, autophagosomes execute autophagy, which delivers intracellular contents to the lysosome. Maturation of endosomes and/or autophagosomes into a lysosome creates an unique acidic environment within the cell for proteolysis and recycling of unneeded cellular components into usable amino acids and other biomolecular building blocks. In the endocytic pathway, gradual maturation of endosomes into a lysosome and acidification of the late endosome are accompanied by vesicle trafficking, protein sorting and targeted degradation of some sorted cargo. Two opposing sorting systems are operating in these processes: the endosomal sorting complex required for transport (ESCRT) supports targeted degradation and the retromer supports retrograde retrieval of certain cargo. The endosomal-lysosomal system is emerging as a central player in a host of neurodegenerative diseases, demonstrating potential roles which are likely to be revealed in pathogenesis and for viable therapeutic strategies. Here we focus on the physiological process of endosomal-lysosomal maturation, acidification and sorting systems along the endocytic pathway, and further discuss relationships between abnormalities in the endosomal-lysosomal system and neurodegenerative diseases, especially Alzheimer's disease (AD).
Small gold nanoparticles (sAuNPs, <10 nm in a core diameter) have been used for drug delivery and cancer therapy due to their high payload to carrier ratio. Information about the amount and location of sAuNPs in cells and tissues is critical to many applications. However, the current detection method (i.e., transmission electron microscopy) for such sAuNPs is limited due to the extensive sample preparation and the limited field of view. Here we use confocal laser scanning microscopy to provide endosome-entrapped sAuNP distributions and to quantify particle uptake into cells. The quantitative capabilities of the system were confirmed by inductively coupled plasma-mass spectrometry, with an observed linear relation between scattering intensity and the initial cellular uptake of sAuNPs using 4 nm and 6 nm core particles.
The summary of the method is:
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This non-invasive imaging strategy provides a tool for label-free real-time tracking and quantification of sAuNPs using a commercially available confocal laser scanning microscope.
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Scattering intensity depends on particle size.
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The linear relation established between scattering intensity and uptaken gold amount enables simultaneous quantitative assessment through simple image analysis.
Depurination has attracted considerable attention since a long time for it is closely related to the damage and repair of nucleic acids. In the present study, depurination using a pool of 30-nt short DNA pieces with various sequences at diverse pH values was analyzed by High Performance Liquid Chromatography (HPLC). Kinetic analysis results showed that non-enzymatic depurination of oligodeoxynucleotides exhibited typical first-order kinetics, and its temperature dependence obeyed Arrhenius' law very well. Our results also clearly showed that the linear relationship between the logarithms of rate constants and pH values had a salient point around pH 2.5. Interestingly and unexpectedly, depurination depended greatly on the DNA sequences. The depurination of poly (dA) was found to be extremely slow, and thymine rich sequences depurinated faster than other sequences. These results could be explained to some extent by the protonation of nucleotide bases. Moreover, two equations were obtained based on our data for predicting the rate of depurination under various conditions. These results provide basic data for gene mutagenesis and nucleic acids metabolism in acidic gastric juice and some acidic organelles, and may also help to rectify some misconceptions about depurination.
Mesenchymal stem cells (MSC) have been identified as having great potential as autologous cell therapeutics to treat traumatic brain injury, spinal injury, as well as neuronal and cardiac ischemic events. All future clinical applications of MSC cell therapies must allow the MSC to be harvested, transfected, and induced to express a desired protein or selection of proteins to have medical benefit. For the full potential of MSC cell therapy to be realized, it is desirable to systematically alter the protein expression of therapeutically beneficial biomolecules in harvested MSC cells with high fidelity in a single transfection event. We have developed a delivery platform based on the use of a solid gold nanoparticle that has been surface modified to produce a fusion containing a zwitterionic, pentapeptide designed from Bax-inhibiting peptide (Ku70) to enhance cellular uptake and a linearized expression vector to induce enhanced expression of brain derived neurotrophic factor (BDNF) in rat derived MSCs. Ku70 is observed to effect >80% transfection following a single treatment of femur bone marrow isolated rat MSCs with efficiencies for the delivery of a 6.6kbp gene on either a AuNP or CdSe/ZnS QD. Gene expression is observed within 4d by optical measurements and secretion observed within 10d by Western Blot analysis. The combination of being able to selectively engineer the NP, co-localize biological agents, and enhance the stability of those agents have provided the strong impetus to utilize this novel class of materials to engineer primary MSCs.
Significance
Previous research has investigated the interactions between linear oligonucleotides and serum nucleases but the emergence of oligonucleotides conjugated to nanoparticles opens up a new avenue of research that has yet to be fully explored. In this work, we probed the degradation of a model small interfering RNA (siRNA) immobilized on gold nanoparticles (AuNPs). We show that serum nucleases are able to recognize and degrade the immobilized siRNA at specific sites within 4 nm of the AuNP surface, which were different than the sites for siRNA free in solution. These data suggest that oligonucleotides immobilized on nanoparticle surfaces must be studied independently from siRNA free in solution due to the potential influence of the local chemical environment of the oligonucleotide–nanoparticle conjugates.
Polycations such as polyethylenimine (PEI) are used in many novel nonviral vector designs and there are continuous efforts to increase our mechanistic understanding of their interactions with cells. Even so, the mechanism of polyplex escape from the endosomal/lysosomal pathway after internalization is still elusive. The "proton sponge " hypothesis remains the most generally accepted mechanism, although it is heavily debated. This hypothesis is associated with the large buffering capacity of PEI and other polycations, which has been interpreted to cause an increase in lysosomal pH even though no conclusive proof has been provided. In the present study, we have used a nanoparticle pH sensor that was developed for pH measurements in the endosomal/lysosomal pathway. We have carried out quantitative measurements of lysosomal pH as a function of PEI content and correlate the results to the "proton sponge " hypothesis. Our measurements show that PEI does not induce change in lysosomal pH as previously suggested and quantification of PEI concentrations in lysosomes makes it uncertain that the "proton sponge " effect is the dominant mechanism of polyplex escape.Molecular Therapy (2012); doi:10.1038/mt.2012.185.
Fiji is a distribution of the popular open-source software ImageJ focused on biological-image analysis. Fiji uses modern software engineering practices to combine powerful software libraries with a broad range of scripting languages to enable rapid prototyping of image-processing algorithms. Fiji facilitates the transformation of new algorithms into ImageJ plugins that can be shared with end users through an integrated update system. We propose Fiji as a platform for productive collaboration between computer science and biology research communities.
Intracellular pH (pH(i)) plays a critical role in the physiological and pathophysiological processes of cells, and fluorescence imaging using pH-sensitive indicators provides a powerful tool to assess the pH(i) of intact cells and subcellular compartments. Here we describe a nanoparticle-based ratiometric pH sensor, comprising a bright and photostable semiconductor quantum dot (QD) and pH-sensitive fluorescent proteins (FPs), exhibiting dramatically improved sensitivity and photostability compared to BCECF, the most widely used fluorescent dye for pH imaging. We found that Förster resonance energy transfer between the QD and multiple FPs modulates the FP/QD emission ratio, exhibiting a >12-fold change between pH 6 and 8. The modularity of the probe enables customization to specific biological applications through genetic engineering of the FPs, as illustrated by the altered pH range of the probe through mutagenesis of the fluorescent protein. The QD-FP probes facilitate visualization of the acidification of endosomes in living cells following polyarginine-mediated uptake. These probes have the potential to enjoy a wide range of intracellular pH imaging applications that may not be feasible with fluorescent proteins or organic fluorophores alone.
A new architecture has been designed by the conjugation of [(18)F]2-fluoro-2-deoxy-D-glucose ((18)F-FDG), gold nanoparticles (AuNPs), and anti-metadherin (Anti-MTDH) antibody which is specific to the metadherin (MTDH) over-expressed on the surface of breast cancer cells. Mannose triflate molecule is used as a precursor for synthesis of (18)F-FDG by nucleophilic fluorination. For the conjugation of (18)F-FDG and AuNPs, cysteamine was first bound to mannose triflate (Man-CA) before synthesizing of (18)F-FDG which has cysteamine sides ((18)FDG-CA). Then, (18)FDG-CA was reacted with HAuCl(4) to obtain AuNPs and with NaBH(4) for reduction of AuNPs. At the end of this procedure, AuNPs were conjugated to (18)F-FDG via disulphide bonds ((18)FDG-AuNP). For the conjugation of Anti-MTDH, 1,1'-carbonyl diimidazol (CDI) was bound to the (18)FDG-AuNP, and Anti-MTDH was conjugated via CDI ((18)FDG-AuNP-Anti-MTDH). This procedure was also performed by using Na(19)F to obtain non-radioactive conjugates ((19)FDG-AuNP-Anti-MTDH). Scanning electron microscopy (SEM) images demonstrated that synthesized particles were in nano sizes. (18)FDG-AuNP-Anti-MTDH conjugate was characterized and used as a model probe containing both radioactive and optical labels together as well as the biological target. The (18)FDG-AuNP-Anti-MTDH conjugate was applied to MCF7 breast cancer cell line and apoptotic cell ratio was found to be increasing from 2% to 20% following the treatment. Hence, these results have promised an important application potential of this conjugate in cancer research.
Psoriasis is a T-cell-mediated skin disease with autoimmune nature that is generally not observed in animals, this lack of a relevant experimental animal model of psoriasis has hindered the investigation of pathogenesis of disease. Application and systemic delivery of small interfering RNAs offer many effective therapeutic advantages for gene regulation in the skin.
In this study, we present an IMQ animal model of psoriasis and designed a safe fusion peptide carrier, spherical nucleic acid gold nanoparticles conjugate, to improve penetration of the siRNA into the cells and skin and their targeting ability to gene regulation. We evaluated the model of psoriasis and EGFR siRNA treatment (as spherical nucleic acid nanoparticles), phenotypically (signs of erythema, scaling, inflammation and thickening), microscopic evaluation of cell proliferation and immunohistochemically evaluation of CD3, CD4, and CD8 markers. Also, we monitored suppression of EGF&EGFR genes after treatment of A431 cells by SNA-NCs. The expression of genes was validated by qRT–PCR in human skin cells.
The results showed that the SNA-NCs were stable and non-toxic. In vitro experiments indicated that EGF&EGFR siRNAs conjugated with spherical nucleic acid gold nanoparticles can significantly reduce gene expression in cells. In vivo experiments showed that the topical application of siRNAs delivered by SNA-NCs through the skin can significantly inhibit the proliferation of cells. Microscopic evaluation of mice back skin and immunohistochemistry process approved Inhibitory effect of SNA-NCs siRNA in the mouse model of psoriasis.
Since the proliferation of T cells was crucial for the development of a psoriatic phenotype. These results demonstrate that topical application of SNA-NCs siRNA may improve psoriatic-like skin lesions by suppressing gene expression and functional activity of T cell production.
By taking advantage of the optical properties of upconversion nanoparticles (UCNPs), we have designed a luminescence ratiometric nanosensor for measuring nitric oxide (NO) in biological fluids, live cells, and tissues. This nanoconjugate consists of a UCNP core with two strong fluorescence emission peaks at 540 and 656 nm as the upconversion fluorophore, NO-reactive rhodamine B-derived molecules (RdMs) encapsulated within the mesopores of the mSiO2 shell, and a β-cyclodextrin (βCD) layer on the exterior of the particle. Reaction of the analyte with the O-phenylenediamine of the RdM induces opening of the spiro-ring and is accompanied by an appearance of a strong rhodamine B (RdB) absorption band between 500~600 nm, which has spectral overlap with the green emission (540 nm) of the UCNPs. This results in an increase in the I656/I540ratio and quantitatively correlates with [NO]. The assay is validated under clean buffer conditions as well as inserum and liver tissue slices obtained from mouse models.
Infectious diseases are a major global threat that accounts for one of the leading causes of global mortality and morbidity. Prompt diagnosis is a crucial first step in the management of infectious threats, which aims to quarantine infected patients to avoid contacts with healthy individuals and deliver effective treatments prior to further spread of diseases. This review article discusses current advances of diagnostic systems using colloidal nanomaterials (e.g., gold nanoparticles, quantum dots, magnetic nanoparticles) for identifying and differentiating infectious pathogens. The challenges involved in the clinical translation of these emerging nanotechnology based diagnostic devices will also be discussed.
Accurate determination of intracellular glucose is very important for exploring its chemical and biological functions in metabolism events of living cells. In this paper, we developed a new non-invasive and highly selective nanokit for intracellular glucose monitoring via two steps recognition. The liposome-based nanokit coencapsulated the aptamer-functionalized gold nanoparticles (AuNPs) and the Shinkai’s receptor together. Upon the proposed nanokit was transfected into living cells, the Shinkai’s receptor could recognize glucose firstly and then changed its conformation to endow aptamers with binding and sensing properties which are not readily accessible otherwise. Then, the binary complexes formed by the intracellular glucose and the Shinkai’s receptor can in situ displace the complementary oligonucleotide of the aptamer on the surface of AuNPs. The fluorophore-labeled aptamer was away from the AuNPs and the fluorescent state switched from “off” to “on”. Through the secondary identification of aptamer, the selectivity of the Shinkai’s receptor could be greatly improved while the intracellular glucose level was assessed by fluorescence signal recovery of aptamer. In the follow-up application, the approach exhibits excellent selectivity and non-invasive for intracellular glucose monitoring under normoxia and hypoxia. To the best of our knowledge, this is the first time that combing advantages of organic receptors and nucleic acids and then realize highly selective monitoring of intracellular glucose via two-steps recognition. We except it will open up new possibilities to integrate devices for diagnosis of various metabolic diseases and insulin delivery.
Gold nanoparticles (AuNPs) show potential for transfecting target cells with small interfering RNA (siRNA), but the influence of key design parameters such as the size and shape of the particle core is incomplete. This paper describes a side-by-side comparison of the in vitro response of U87 glioblastoma cells to different formulations of siRNA-conjugated gold nanoconstructs targeting the expression of isocitrate dehydrogenase 1 (IDH1) based on 13-nm spheres, 50-nm spheres, and 40-nm stars. 50-nm spheres and 40-nm stars showed much higher uptake efficiency compared to 13-nm spheres. Confocal fluorescence microscopy showed that all three formulations were localized in the endosomes at early incubation times (2 h) but that after 24 h, 50-nm spheres and 40-nm stars were neither in endosomes nor lysosomes while 13-nm spheres remained in endosomes. Transmission electron microscopy images revealed that the 13-nm spheres were enclosed and dispersed within endocytic vesicles while 50-nm spheres and 40-nm stars were aggregated, and some of these NPs were outside of endocytic vesicles. In our comparison of nanoconstructs with different sizes and shapes, while holding siRNA surface density and nanoparticle concentration constant, we found that larger particles (50-nm spheres and 40-nm stars) showed higher potential as carriers for the delivery of siRNA.
Measurement of changes of pH at various intracellular compartments has potential to solve questions concerning the processing of endocytosed material, regulation of the acidification process and also, acidification of vesicles destined for exocytosis. To monitor these events the nanosized optical pH probes need to provide ratiometric signals in the optically transparent biological window, target to all relevant intracellular compartments, and to facilitate imaging at subcellular resolution without interference from biological matrix. To meet these criteria we sensitize the surface conjugated pH sensitive indicator via an upconversion process utilizing an energy transfer from the nanoparticle to the indicator. Live cells were imaged with a scanning confocal microscope equipped with a low energy 980 nm laser excitation, that facilitated high resolution and penetration depth into the specimen, and low phototoxicity needed for long-term imaging. Our upconversion nanoparticle resonance energy transfer based sensor with polyethylenimine-coating provides high colloidal stability, enhanced cellular uptake and distribution across cellular compartments. This distribution was modulated with membrane integrity perturbing treatment that resulted into total loss of lysosomal compartments and a dramatic pH shift of endosomal compartments. These nanoprobes are well suited for detection of pH changes in in vitro models with high biological background fluorescence and in in vivo applications, e.g. for the bioimaging of small animal models.
The present article reports the development of nanohybrid comprising of anionic carbon dots (ACD) protected gold nanoparticle (GNP). ACD directly cap GNP through its anionic surface functionalization leading to the formation of stable aqueous GNP dispersion. This newly developed ACD-GNP nanohybrid has been thoroughly characterized by different spectroscopic and microscopic techniques. This nanohybrid is successfully employed towards the selective sensing of glutathione (GSH). The mechanism of GSH sensing by this nanosensor is based on the GSH triggered displacement of fluorescent indicator ACD from the GNP surface. Upon capping GNP, intrinsic fluorescence of ACD gets quenched. Addition of GSH displaces the fluorescent indicator ACD from GNP surface and restores the fluorescence signal of ACD. This nanosensor exhibits very high selectivity as well as sensitivity towards glutathione over the other biothiols and can detect as low as 6 nM of GSH. More importantly, selective imaging of the cancer cells over the non-cancerous cells was achieved by this ACD-GNP hybrid implying its potential applications in biosensing as well as in cancer diagnosis.
Ribonucleic acids (RNAs) are key components in many cellular processes such as cell division, differentiation, growth, aging, and death. RNA spherical nucleic acids (RNA-SNAs), which consist of dense shells of double-stranded RNA on nanoparticle surfaces, are powerful and promising therapeutic modalities because they confer advantages over linear RNA such as high cellular uptake and enhanced stability. Due to their three-dimensional shell of oligonucleotides, SNAs, in comparison to linear nucleic acids, interact with the biological environment in unique ways. Herein, the modularity of the RNA-SNA is used to systematically study structure-function relationships in order to understand how the oligonucleotide shell affects interactions with a specific type of biological environment, namely, one that contains serum nucleases. We use a combination of experiment and theory to determine the key architectural properties (i.e., sequence, density, spacer moiety, and backfill molecule) that affect how RNA-SNAs interact with serum nucleases. These data establish a set of design parameters for SNA architectures that are optimized in terms of stability.
Optical ruler methods employing multiple fluorescent labels offer great potential for correlating distances among several sites, but are generally limited to inter-label distances under 10 nm and suffer from complications due to spectral overlap. Here we demonstrate a multi-color surface energy transfer (McSET) technique able to triangulate multiple points on a biopolymer, allowing for analysis of global structure in complex biomolecules. McSET couples the competitive energy transfer pathways of Förster Resonance Energy Transfer (FRET) with gold-nanoparticle mediated Surface Energy Transfer (SET) in order to correlate systematically labeled points on the structure at distances greater than 10 nm and with reduced spectral overlap. To demonstrate the McSET method, the structures of a linear B-DNA and a more complex folded RNA ribozyme were analyzed within the McSET mathematical framework. The improved multicolor optical ruler method takes advantage of the broad spectral range and distances achievable when using a gold nanoparticle as the lowest energy acceptor. The ability to report distance information simultaneously across multiple length scales, short range (10-50 Å), mid-range (50-150 Å) and long-range (150-350 Å), distinguishes this approach from other multicolor energy transfer methods.
Great challenges in investigating the release of drug in complex cellular microenvironments necessitate the development of stimuli-responsive drug delivery systems with real-time monitoring capability. In this work, a smart drug nanocarrier based on fluorescence resonance energy transfer (FRET) is fabricated by capping graphene quantum dots (GQDs, the acceptor) onto fluorescent mesoporous silica nanoparticles (FMSNs, the donor) via ATP aptamer for real-time monitoring of ATP-triggered drug release. Under extracellular conditions, the fluorescence of FMSNs remains "off" state in low ATP level which is unable to trigger the release of drug. Once specifically recognized and internalized into the target tumor cells by AS1411 aptamer, in the ATP-rich cytoplasm, the conformation switch of the ATP aptamer causes the shedding of the GQDs from the nanocarriers, leading to the release of the loaded drugs and consequently severe cytotoxicity. Simultaneously, the fluorescence of FMSNs turns "on" along with the dissociation of GQDs, which allows real-time monitoring the release of drug from the pores. Such a drug delivery system features high specificity of dual-target recognition with AS1411 and ATP aptamer as well as high sensitivity of FRET based monitoring strategy. Thus, the proposed multifunctional ATP triggered FRET-nanocarriers will find potential applications for versatile drug-release monitoring, efficient drug transport, and targeted cancer therapeutics.
We designed a new ratiometric fluorescent nanoprobe for sensing pH values in living cells. Briefly, the nanoprobe consists of a gold nanoparticle (AuNP), short single-stranded oligonucleotides, and dual-fluorophore-labeled i-motif sequences. The short oligonucleotides are designed to bind with the i-motif sequences, and immobilized on AuNP surface via Au-S bond. At neutral pH, the dual fluorophores are separated, resulting in very low fluorescence resonance energy transfer (FRET) efficiency. At acidic pH, the i-motif strands fold into quadruplex structure and leave AuNP away, bringing the dual fluorophores into close proximity, resulting in high FRET efficiency, which could be used as a signal for pH sensing. The nanoprobe possesses abilities of cellular transfection, enzymatic protection, fast response and quantitative pH detection. The in vitro and intracellular applications of the nanoprobe were demonstrated, which showed excellent response in the physiological pH range. Furthermore, our experimental results suggested that the nanoprobe showed excellent spatial and temporal resolution in living cells. We think that the ratiometric sensing strategy could potentially be applied to create a variety of new multicolor sensors for intracellular detection.
Nanosensors offer significant advantages over earlier methods of metabolite measurements in live cells, which previously relied on loading cells with free dyes. A nanosensor sensitive to reactive oxygen species (ROS) has been developed by incorporating the ROS-sensitive fluorescent probe dihydrorhodamine 123 along with a reference dye into a polyacrylamide matrix. A triple-labeled nanosensor with two pH-sensitive fluorophores and a reference fluorophore for a very broad sensitivity range has recently been developed. Measurements of metabolites in cells with optical sensors can be performed by various techniques, which utilize one or more light sources for excitation and one or more detectors for monitoring photon emission. Flow cytometry has the potential to analyze very large cell populations on a single-cell level, resulting in large amounts of statistical material; however, as with spectroscopy, subcellular distribution of the sensor is not considered and the fluorescence within a cell is averaged. Calibration of the sensor has to be prepared on the same instrument where the cell measurements are performed. The simplest calibration method is to perform in vitro calibration, in which the response of the indicator is measured as a function of known metabolite concentration in solutions.
Recently, ratiometric pH nanosensors have emerged as a robust tool for the fluorescence sensing and imaging, but there is no report of ratiometric sensors based on hyperbranched polymers for intracellular pH sensing. Herein we describe the first example of hyperbranched polymer-based tunable fluorescent pH nanosensor with aggregation-induced emission activity, which exhibits great potential for ratiometric sensing of intracellular pH. These polymer nanoparticles can selectively accumulate in the acidic organelles of living cells by endocytosis process, and no obvious cytotoxicity was observed. The quantitative analysis of the intracellular pH values in HeLa cells was successfully conducted based on this new sensing platform. This platform provides a new choice for future developments of ratiometric fluorescent nanosensors, not only targeting protons, but also a variety of other analytes of biological interest, such as metal ions, anions, and other biomolecules.
The absorbance of the organic water tracer compound fluorescein is known to be pH dependent but differences between the reported PKa values make it difficult to predict these absorbance changes. A new pKa determination method, which incorporated activity corrections, was used to calculate the pKa values of fluorescein. Several published pKa values were re-evaluated and were in agreement once activity corrections were applied. WaterSA Vol.28(4) 2002: 395-402
Nanometal surface energy transfer (NSET) techniques on gold nanoparticles (AuNPs) have become an essential tool in molecular biophysics to identify structural details at long-range donor-acceptor distances. The NSET mechanism is well described, but it has been suggested that the use of large AuNPs in NSET may manipulate natural biomolecular function. If, in fact, such non-specific interactions with the AuNP surface can be quantified or contained, then NSET may offer more potential in tracking biomolecular folding than the most comprehensive methods in conformer determination (X-ray crystallography, NMR, EPR). Here we describe an NSET ruler capable of tracking Hybrid-2 telomere quadruplex folding and we demonstrate that nucleic acid appendage to AuNPs up to 10nm in diameter does not manipulate biomolecular function. The quadruplex folding of Hybrid-2 sequences was tracked by monitoring the emission of a DY680 dye on selected basepairs in the telomere sequence when appended to the surface of AuNPs (5-10nm). Emission-derived distances extracted from NSET theory correlate well to reported NMR structures of the antiparallel quadruplex. Moreover, NSET theory calculates identical donor-acceptor distal points between DY680 and all sizes of AuNPs, indicating that the AuNP tether is not dominant or disruptive towards nucleic acid folding. This article is protected by copyright. All rights reserved.
This article is protected by copyright. All rights reserved.
Recent advances in cell transfection have suggested that delivery of a gene on a gold nanoparticle (AuNP) can enhance transfection efficiency. The mechanism of transfection is poorly understood, particularly when the gene is appended to an AuNP, as expression of the desired exogenous protein is dependent not only on the efficiency of the gene being taken into the cell, but also on efficient endosomal escape and cellular processing of the nucleic acid. Design of a multicolor surface energy transfer (McSET) molecular beacon by independently dye labeling a linearized plasmid and short duplex DNA (sdDNA) appended to an AuNP, allows spatiotemporal profiling the transfection events providing insight into package uptake, disassembly, and final plasmid expression. Delivery of the AuNP construct encapsulated in Lipofectamine2000® is monitored in Chinese hamster ovary cells using live-cell confocal microscopy. The McSET beacon signals the location and timing of the AuNP release and endosomal escape events for the plasmid and the sdDNA discretely, which are correlated with plasmid transcription by fluorescent protein expression within the cell. It is observed that delivery of the construct leads to endosomal release of the plasmid and sdDNA from the AuNP surface at different rates, prior to endosomal escape. Slow cytosolic diffusion of the nucleic acids is believed to be the limiting step for transfection, impacting the time dependent expression of protein. The overall protein expression yield is enhanced when delivered on an AuNP, possibly due to better endosomal escape or lower degradation prior to endosomal escape.
Cysteine is one of the least abundant amino acids, yet it is frequently found as a highly conserved residue within functional (regulatory, catalytic or binding) sites in proteins. It is the unique chemistry of the thiol or thiolate group of cysteine that imparts functional sites with their specialized properties (e.g., nucleophilicity, high affinity metal binding, and/or ability to form disulfide bonds). Highlighted in this review are some of the basic biophysical and biochemical properties of cysteine groups and the equations that apply to them, particularly with respect to pKa and redox potential. Also summarized are the types of low molecular weight thiols present in high concentrations in most cells, as well as the ways in which modifications of cysteinyl residues can impart or regulate molecular functions important to cellular processes including signal transduction.
Spherical nucleic acid (SNA) nanoparticle conjugates are a class of bionanomaterials that are extremely potent in many biomedical applications. Their unique ability to enter multiple mammalian cell types as single-entity agents arises from their novel three-dimensional architecture, which consists of a dense shell of highly oriented oligonucleotides chemically attached typically to a gold nanoparticle core. This architecture allows SNAs to engage certain cell surface receptors to facilitate entry. Here, we report studies aimed at determining the intracellular fate of SNAs and the trafficking events that occur inside C166 mouse endothelial cells after cellular entry. We show that SNAs traffic through the endocytic pathway into late endosomes and reside there for up to 24 h after incubation. Disassembly of oligonucleotides from the nanoparticle core is observed 16 h after cellular entry, most likely due to degradation by enzymes such as DNase II localized in late endosomes. Our observations point to these events being likely independent of core composition and treatment conditions, and they do not seem to be particularly dependent upon oligonucleotide sequence. Significantly and surprisingly, the SNAs do not enter the lysosomes under the conditions studied. To independently track the fate of the particle core and the fluorophore-labeled oligonucleotides that comprise its shell, we synthesized a novel class of quantum dot SNAs to determine that as the SNA structures are broken down over the 24 h time course of the experiment, the oligonucleotide fragments are recycled out of the cell while the nanoparticle core is not. This mechanistic insight points to the importance of designing and synthesizing next-generation SNAs that can bypass the degradation bottleneck imposed by their residency in late endosomes, and it also suggests that such structures might be extremely useful for endosomal signaling pathways by engaging receptors that are localized within the endosome.
With advances in therapeutic science, apart from drugs, newer bioactive moieties like oligonucleotides, proteins, peptides, enzymes and antibodies are constantly being introduced for the betterment of therapeutic efficacy. These moieties have intracellular components of the cells like
cytoplasm and nucleus as one of their pharmacological sites for exhibiting therapeutic activity. Despite their promising efficacy, their intracellular bioavailability has been critically hampered leading to failure in the treatment of numerous diseases and disorders. The endosomal uptake pathway
is known to be a rate-limiting barrier for such systems. Bioactive molecules get trapped in the endosomal vesicles and degraded in the lysosomal compartment, necessitating the need for effective strategies that facilitate the endosomal escape and enhance the cytosolic bioavailability of bioactives.
Microbes like viruses and bacteria have developed their innate mechanistic tactics to translocate their genome and toxins by efficiently penetrating the host cell membrane. Understanding this mechanism and exploring it further for intracellular delivery has opened new avenues to surmount the
endosomal barrier. These strategies include membrane fusion, pore formation and proton sponge effects. On the other hand, progress in designing a novel smart polymeric carrier system that triggers endosomal escape by undergoing modulations in the intracellular milieu has further led to an
improvement in intracellular delivery. These comprise pH, enzyme and temperature-induced modulators, synthetic cationic lipids and photo-induced physical disruption. Each of the aforementioned strategies has its own unique mechanism to escape the endosome. This review recapitulates the numerous
strategies designed to surmount the bottleneck of endosomal escape and thereby achieve successful intracellular uptake of bioactives.
Understanding the behavior of multifunctional colloidal nanoparticles capable of biomolecular targeting remains a fascinating challenge in materials science with dramatic implications in view of a possible clinical translation. In several circumstances, assumptions on structure-activity relationships have failed in determining the expected responses of these complex systems in a biological environment. The present Review depicts the most recent advances about colloidal nanoparticles designed for use as tools for cellular nanobiotechnology, in particular, for the preferential transport through different target compartments, including cell membrane, cytoplasm, mitochondria, and nucleus. Besides the conventional entry mechanisms based on crossing the cellular membrane, an insight into modern physical approaches to quantitatively deliver nanomaterials inside cells, such as microinjection and electro-poration, is provided. Recent hypotheses on how the nanoparticle structure and functionalization may affect the interactions at the nano-bio interface, which in turn mediate the nanoparticle internalization routes, are highlighted. In addition, some hurdles when this small interface faces the physiological environment and how this phenomenon can turn into different unexpected responses, are discussed. Finally, possible future developments oriented to synergistically tailor biological and chemical properties of nanoconjugates to improve the control over nanoparticle transport, which could open new scenarios in the field of nanomedicine, are addressed.
The aggregation of gold nanoparticles (Au NPs) in cell media is a common phenomenon that can influence NP-cell interactions. Here, we control Au NP aggregation in cell media and study the impact of Au NP aggregation on human dermal fibroblast (HDF) cells. By first adding Au NPs to fetal bovine serum (FBS) and then subsequently to a buffer, aggregation can be avoided. Aggregation of Au NPs also can be avoided by coating Au NPs with other biomolecules such as lipids. The aggregation state of the Au NPs influences cellular toxicity and Au NP uptake: non-aggregated cationic Au NPs are four-fold less toxic to HDF cells than aggregated cationic Au NPs, and the uptake of non-aggregated anionic citrate Au NPs is three orders of magnitude less than that of aggregated citrate Au NPs. Upon uptake of Au NPs, cellular F-actin fiber formation is disrupted and actin dots are predominant. When lipid-coated Au NPs are doped with a fluorescent lipid (F-lipid) and incubated with HDF cells, the fluorescence from the F-lipid was found throughout the cell, showing that lipids can dissociate from the Au NP surface upon entering the cell.
The interaction of a fluorescent molecule with a gold nanoparticle is complex and can lead to excited-state enhancement or quenching. Many attempts have been made to explain the observed interaction when in close proximity to the metal surface; yet no single model has been capable of explaining the observations. In this work, we show that by accurately describing the interaction in terms of an induced image dipole modified within the gold nanoparticle by the size-dependent changes in absorptivity and dielectric constant, the oscillator interaction can be fully described in terms of a surface-moderated interaction. Comparison of experimental and theoretical data confirms the validity of the model for a selected range of separation distances, nanoparticle radii, and fluorescent molecule selection. The results of the study illustrate the importance of nonradiative pathways for modifying the decay of a fluorescent molecule by coupling to the image dipole, thus providing a firm understanding of the reported variance in behavior for an emitting species in close proximity to nanometal surfaces. A more significant impact of the results is the ability to apply nanometal surface energy transfer methods as a molecular ruler to probe physical questions at much greater distances (>400 Å) than previously achievable.
A ratiometric fluorescent pH sensor based on CdSe/CdZnS nanocrystal quantum dots (NCs) has been designed for biological pH ranges. The construct is formed from the conjugation of a pH dye (SNARF) to NCs coated with a poly(amido amine) (PAMAM) dendrimer. The sensor exhibits a well-resolved ratio response at pH values between 6 and 8 under linear or two-photon excitation, and in the presence of a 4% bovine serum albumin (BSA) solution.
SUMMARYA method to measure the degree of co‐localization of objects in confocal dual‐colour images has been developed. This image analysis produced two coefficients that represent the fraction of co‐localizing objects in each component of a dual‐channel image. The generation of test objects with a Gaussian intensity distribution, at well‐defined positions in both components of dual‐channel images, allowed an accurate investigation of the reliability of the procedure. To do that, the co‐localization coefficients were determined before degrading the image with background, cross‐talk and Poisson noise. These synthesized sources of image deterioration represent sources of deterioration that must be dealt with in practical confocal imaging, namely dark current, non‐specific binding and cross‐reactivity of fluorescent probes, optical cross‐talk and photon noise. The degraded images were restored by filtering and cross‐talk correction. The co‐localization coefficients of the restored images were not significantly different from those of the original undegraded images. Finally, we tested the procedure on images of real biological specimens. The results of these tests correspond with data found in the literature. We conclude that the co‐localization coefficients can provide relevant quantitative information about the positional relation between biological objects or processes.
Aptamers are single-stranded oligonucleotides synthesized through an in vitro selection and amplification process that involves systematic evolution of ligands by exponential enrichment. Based on their high binding affinity and specificity towards other molecules, aptamers generated during the final rounds of selection can be utilized in applications ranging from biosensing to diagnostics and therapeutics. Meanwhile, advances in nanotechnology have led to new and improved materials for biomedical applications. Specifically, nanoparticles can readily interact with both intra- and extra-cellular biomolecules to yield improved signal amplification and target recognition. By combining both technologies, aptamer-conjugated nanoparticles, especially gold nanoparticles (Apt-AuNPs), offer great promise for applications in bioanalysis and biomedicine, including early diagnosis and drug delivery. This review summarizes recent methodologies that have increased the application of Apt-AuNPs in biomedicine, and discusses the potential of Apt-AuNPs in bioanalysis.
We report stimuli-responsive core-satellite assemblies of binary gold nanoparticles, linked by i-motif DNA, for live cell plasmonic imaging of pH changes in the endocytic pathway.
Polycations having a high buffering capacity in the endosomal pH range, such as polyethylenimine (PEI), are known to be efficient at delivering nucleic acids by overcoming lysosomal sequestration possibly through the proton sponge effect, although other mechanisms such as membrane disruption arising from an interaction between the polycation and the endosome/lysosome membrane, have been proposed. Chitosan is an efficient delivery vehicle for nucleic acids, yet its buffering capacity has been thought to be significantly lower than that of PEI, suggesting that the molecular mechanism responsible for endolysosomal escape was not proton sponge based. However, previous comparisons of PEI and chitosan buffering capacity were performed on a mass concentration basis instead of a charge concentration basis, the latter being the most relevant comparison basis because polycation-DNA complexes form at ratios of charge groups (amine to phosphate), rather than according to mass. We hypothesized that chitosan has a high buffering capacity when compared to PEI on a molar basis and could therefore possibly mediate endolysosomal release through the proton sponge effect. In this study, we examined the ionization behavior of chitosan and chitosan-DNA complexes and compared to that of PEI and polylysine on a charge concentration basis. A mean field theory based on the use of the Poisson-Boltzmann equation and an Ising model were also applied to model ionization behavior of chitosan and PEI, respectively. We found that chitosan has a higher buffering capacity than PEI in the endolysosomal pH range, while the formation of chitosan-DNA complexes reduces chitosan buffering capacity because of the negative electrostatic environment of nucleic acids that facilitates chitosan ionization. These data suggest that chitosans have a similar capacity as PEI to mediate endosomal escape through the proton sponge effect, possibly in a manner which depends on the presence of excess chitosan.
A new advance in cell transfection protocol using a bimodal nanoparticle agent to selectively manipulate protein expression levels within mammalian cells is demonstrated. The nanoparticle based transfection approach functions by controlled release of gene regulatory elements from a 6nm AuNP surface. Unique control over release of the regulatory elements reflects the covalent appendage and subsequent packaging of the nucleic acid based oligomers resulting in enhanced endosomal stability and osmatocu pressure enhanced release of the genetic modifiers once the agent is transfected leading to the ability to induce endogenous protein knockdown simultaneously with exogenous protein expression. The use of fluorescent proteins as the endogenous and exogenous signals for protein expression allow the efficiency of co-delivery of siRNA for GFP knockdown and a dsRed-express linearized plasmid for induction to be optically analyzed in CRL-2794, a human kidney cell line expressing an unstable green fluorescent protein. Delivery of the bimodal nanoparticle in cationic liposomes results in 20% GFP knockdown within 24h of delivery and continues exhibiting knockdown for up to 48h for the bimodal agent. Simultaneous dsRed expression is observed to initiate within the same timeframe with expression levels reaching 34% after 25 days although cells have divided approximately twenty times, implying daughter cell transfection has occurred. Fluorescence cell sorting results in a stable colony, as demonstrated by Western blot analysis. The simultaneous delivery of siRNA and linearized plasmid DNA on the surface of a single nanocrystal provides a unique method for defini-tive genetic control within a single cell and leads to a very efficient cell transfection protocol.
Spherical nucleic acid (SNA) constructs are promising new single entity gene regulation materials capable of both cellular transfection and gene knockdown, but thus far are promiscuous structures, exhibiting excellent genetic but little cellular selectivity. In this communication, we describe a strategy to impart targeting capabilities to these constructs through noncovalent functionalization with a complementary antibody-DNA conjugate. As a proof-of-concept, we designed HER2-targeting SNAs and demonstrated that such structures exhibit cell type selectivity in terms of their uptake, and significantly greater gene knockdown in cells overexpressing the target antigen as compared to the analogous antibody-free and off-target materials.
The acidic dissociation constants of a number of thiols have been determined and collated with those already recorded in the literature.
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A study was conducted to demonstrate the use of gold nanoparticles (AuNP) in chemical and biological sensing. AuNPs possessed distinct physical and chemical attributes that made them excellent scaffolds for the fabrication of novel chemical and biological sensors. AuNPs were synthesized in a straightforward manner and made highly stable, while possessing unique optoelectronic properties and providing high surface-to-volume ratio with excellent biocompatibility using appropriate ligands. These synthetic routes and properties of AuNPs made them excellent probes for different sensing strategies. A variety of other ligands had been used to passivate and functionalize AuNPs, while most AuNP functionalization had been done using thiol or thiolated ligands.
We report the development of the multiplexed nanoflare, a nanoparticle agent that is capable of simultaneously detecting two distinct mRNA targets inside a living cell. These probes are spherical nucleic acid (SNA) gold nanoparticle (Au NP) conjugates consisting of densely packed and highly oriented oligonucleotide sequences, many of which are hybridized to a reporter with a distinct fluorophore label and each complementary to its corresponding mRNA target. When multiplexed nanoflares are exposed to their targets, they provide a sequence specific signal in both extra- and intracellular environments. Importantly, one of the targets can be used as an internal control, improving detection by accounting for cell-to-cell variations in nanoparticle uptake and background. Compared to single-component nanoflares, these structures allow one to determine more precisely relative mRNA levels in individual cells, improving cell sorting and quantification.