Labeling of cells with nanoparticles for living detection is of interest to various biomedical applications. In this study, novel fluorescent/magnetic nanoparticles were prepared and used in high-efficient cellular imaging. The nanoparticles coated with the modified chitosan possessed a magnetic oxide core and a covalently attached fluorescent dye. We evaluated the feasibility and efficiency in labeling cancer cells (SMMC-7721) with the nanoparticles. The nanoparticles exhibited a high affinity to cells, which was demonstrated by flow cytometry and magnetic resonance imaging. The results showed that cell-labeling efficiency of the nanoparticles was dependent on the incubation time and nanoparticles’ concentration. The minimum detected number of labeled cells was around 104 by using a clinical 1.5-T MRI imager. Fluorescence and transmission electron microscopy instruments were used to monitor the localization patterns of the magnetic nanoparticles in cells. These new magneto-fluorescent nanoagents have demonstrated the potential for future medical use.
Magnetic nanoparticle; Fluorescence; Chitosan; Magnetic resonance imaging
As applications of nanoparticles in medical imaging and biomedicine rapidly expand, the interactions of nanoparticles with living cells have become an area of active interest. For example, intracellular trafficking of nanoparticles – an important part of cell-nanoparticle interaction, has been well studied using plasmonic nanoparticles and optical or optics-based techniques due to the change in optical properties of the nanoparticle aggregates. However, magnetic nanoparticles, despite their wide range of clinical applications, do not exhibit plasmonic-resonant properties and therefore their intracellular aggregation cannot be detected by optics-based imaging techniques. In this study, we investigated the feasibility of a novel imaging technique – pulsed magneto-motive ultrasound (pMMUS), to identify intracellular trafficking of endocytosed magnetic nanoparticles. In pulsed magneto-motive ultrasound imaging a focused, high intensity, pulsed magnetic field is used to excite the cells labeled with magnetic nanoparticles, and ultrasound imaging is then used to monitor the mechanical response of the tissue. We demonstrated previously that clusters of magnetic nanoparticles amplify the pMMUS signal in comparison to signal from individual nanoparticles. Here we further demonstrate that pMMUS imaging can identify interaction between magnetic nanoparticles and living cells, i.e. intracellular aggregation of nanoparticles within the cells. The results of our study suggest that pMMUS imaging can not only detect the presence of magnetic nanoparticles but also provides information about their intracellular trafficking non-invasively and in real-time.
Pulsed magneto-motive ultrasound imaging; superparamagnetic iron-oxide nanoparticles; macrophage; endocytosis; intracellular trafficking
Nano-sized particles are widely regarded as a tool to study biologic events at the cellular and molecular levels. However, only some imaging modalities can visualize interaction between nanoparticles and living cells. We present a new technique, pulsed magneto-motive ultrasound imaging, which is capable of in vivo imaging of magnetic nanoparticles in real time and at sufficient depth. In pulsed magneto-motive ultrasound imaging, an external high-strength pulsed magnetic field is applied to induce the motion within the magnetically labeled tissue and ultrasound is used to detect the induced internal tissue motion. Our experiments demonstrated a sufficient contrast between normal and iron-laden cells labeled with ultrasmall magnetic nanoparticles. Therefore, pulsed magneto-motive ultrasound imaging could become an imaging tool capable of detecting magnetic nanoparticles and characterizing the cellular and molecular composition of deep-lying structures.
Thrombosis underlies numerous life-threatening cardiovascular syndromes. Development of thrombosis-specific molecular imaging agents to detect and monitor thrombogenesis and fibrinolysis in vivo could improve the diagnosis, risk stratification, and treatment of thrombosis syndromes. To this end, we have synthesized efficient multimodal nanoagents targeted to two different constituents of thrombi, namely, fibrin and activated factor XIII. These agents are targeted via the conjugation of peptide-targeting ligands to the surface of fluorescently labeled magnetic nanoparticles. As demonstrated by in vitro and in vivo studies, both nanoagents possess high affinities for thrombi, and enable mutimodal fluorescence and magnetic resonance imaging.
The purpose of this study was to investigate the downregulation of mRNA expression of vascular endothelial growth factor (VEGF) by triamcinolone acetonide acetate (TAA)-loaded chitosan nanoparticles in human retinal pigment epithelial cells.
TAA-loaded deoxycholic acid-modified chitosan (TAA/DA-Chit) nanoparticles were prepared via a self-assembly mechanism, and their morphology and zeta potential were examined by transmission electron microscopy and zeta potential analysis, respectively. DA-Chit and TAA/DA-Chit nanoparticle toxicity was evaluated using a Cell Counting Kit-8 assay. The efficiency of cellular uptake was determined using fluorescein isothiocyanate-labeled DA-Chit nanoparticles, in place of TAA/DA-Chit nanoparticles, assessed by both inverted fluorescence microscopy and flow cytometry. Downregulation of VEGF mRNA expression by TAA/DA-Chit nanoparticles was further investigated by real-time reverse transcription polymerase chain reaction (RT-PCR) assay of the treated human retinal pigment epithelial cells.
TAA/DA-Chit nanoparticles were prepared with a TAA-loading capacity in the range of 12%–82%, which increased the water solubility of TAA from 0.3 mg/mL to 2.1 mg/mL. These nanoparticles showed oblate shapes 100–550 nm in size in transmission electron microscopic images and had positive zeta potentials. The Cell Counting Kit-8 assay indicated that the DA-Chit and TAA/DA-Chit nanoparticles had no toxicity and low toxicity, respectively, to human retinal pigment epithelial cells. Fluorescein isothiocyanate-labeled DA-Chit nanoparticle uptake by human retinal pigment epithelial cells was confirmed by inverted fluorescence microscopy and flow cytometry. Real-time RT-PCR assay showed that the VEGF mRNA level decreased after incubation of human retinal pigment epithelial cells with TAA/DA-Chit nanoparticles.
TAA/DA-Chit nanoparticles had a downregulating effect on VEGF mRNA expression in human retinal pigment epithelial cells and low cytotoxicity, which might be beneficial characteristics for the development of future treatment for diabetic retinopathy.
chitosan; nanoparticle; triamcinolone acetonide acetate; human retinal pigment epithelial cells; vascular endothelial growth factor; mRNA
Functionalization of monodisperse superparamagnetic magnetite (Fe3O4) nanoparticles for cell specific targeting is crucial for cancer diagnostics and therapeutics. Targeted magnetic nanoparticles can be used to enhance the tissue contrast in magnetic resonance imaging (MRI), to improve the efficiency in anticancer drug delivery, and to eliminate tumor cells by magnetic fluid hyperthermia. Herein we report the nucleus-targeting Fe3O4 nanoparticles functionalized with protein and nuclear localization signal (NLS) peptide. These NLS-coated nanoparticles were introduced into the HeLa cell cytoplasm and nucleus, where the particles were monodispersed and non-aggregated. The success of labeling was examined and identified by fluorescence microscopy and MRI. The work demonstrates that monodisperse magnetic nanoparticles can be readily functionalized and stabilized for potential diagnostic and therapeutic applications.
cell imaging; magnetic resonance imaging; magnetite; nanoparticles
Conjugated polymer nanoparticles are formed by precipitation of highly fluorescent conjugated polymers to form small nanoparticles with extremely bright fluorescence. We characterized cellular uptake and cytotoxicity of 18 ± 5 nm PFBT conjugated polymer nanoparticles in J774A.1 cells. Significant nanoparticle uptake was observed, indicating efficient nanoparticle entry into cells, even for short (1 h) incubations. The high fluorescence of these nanoparticles allows extremely low loading concentrations; PFBT nanoparticle fluorescence in cells could be detected with loading concentrations of 155 pM (270 ppb). Cellular uptake slows at low temperature, consistent with endocytic entry. Nanoparticles colocalize with Texas Red dextran and are trafficked to lysosomes, as demonstrated by the location of nanoparticle fluorescence in perinuclear organelles that also stain with an anti-LAMP-1 antibody. Inhibition of uptake by phosphoinositide 3-kinase inhibitors implicates macropinocytosis as the operative endocytic mechanism. No significant cytotoxic or inflammatory effects could be observed, making PFBT nanoparticles attractive probes for live cell imaging.
Intracellular; imaging; cytotoxicity; macropinocytosis; fluorescence; probe
Magnetic nanoparticles are promising molecular imaging agents due to their relative high relaxivity and the potential to modify surface functionality to tailor biodistribution. In this work we describe the synthesis of magnetic nanoparticles using organic solvents with organometallic precursors. This method results in nanoparticles that are highly crystalline, and have uniform size and shape. The ability to create a monodispersion of particles of the same size and shape results in unique magnetic properties that can be useful for biomedical applications with MR imaging. Before these nanoparticles can be used in biological applications, however, means are needed to make the nanoparticles soluble in aqueous solutions and the toxicity of these nanoparticles needs to be studied.
We have developed two methods to surface modify and transfer these nanoparticles to the aqueous phase using the biocompatible co-polymer, Pluronic F127. Cytotoxicity was found to be dependent on the coating procedure used. Nanoparticle effects on a cell-culture model was quantified using concurrent assaying; a LDH assay to determine cytotoxicity and an MTS assay to determine viability for a 24 hour incubation period. Concurrent assaying was done to insure that nanoparticles did not interfere with the colorimetric assay results.
This report demonstrates that a monodispersion of nanoparticles of uniform size and shape can be manufactured. Initial cytotoxicity testing of new molecular imaging agents need to be carefully constructed to avoid interference and erroneous results.
MRI; molecular imaging; nanoparticles; superparamagnetic agents; cytotoxicity; Colorimetric Assay; Pluronics
In recent years, near-infrared fluorescence (NIRF)-labeled iron nanoparticles have been synthesized and applied in a number of applications, including the labeling of human cells for monitoring the engraftment process, imaging tumors, sensoring the in vivo molecular environment surrounding nanoparticles and tracing their in vivo biodistribution. These studies demonstrate that NIRF-labeled iron nanoparticles provide an efficient probe for cell labeling. Furthermore, the in vivo imaging studies show excellent performance of the NIR fluorophores. However, there is a limited selection of NIRF-labeled iron nanoparticles with an optimal wavelength for imaging around 800 nm, where tissue autofluorescence is minimal. Therefore, it is necessary to develop additional alternative NIRF-labeled iron nanoparticles for application in this area.
This study manufactured 12-nm DMSA-coated Fe3O4 nanoparticles labeled with a near-infrared fluorophore, IRDye800CW (excitation/emission, 774/789 nm), to investigate their applicability in cell labeling and in vivo imaging. The mouse macrophage RAW264.7 was labeled with IRDye800CW-labeled Fe3O4 nanoparticles at concentrations of 20, 30, 40, 50, 60, 80 and 100 μg/ml for 24 h. The results revealed that the cells were efficiently labeled by the nanoparticles, without any significant effect on cell viability. The nanoparticles were injected into the mouse via the tail vein, at dosages of 2 or 5 mg/kg body weight, and the mouse was discontinuously imaged for 24 h. The results demonstrated that the nanoparticles gradually accumulated in liver and kidney regions following injection, reaching maximum concentrations at 6 h post-injection, following which they were gradually removed from these regions. After tracing the nanoparticles throughout the body it was revealed that they mainly distributed in three organs, the liver, spleen and kidney. Real-time live-body imaging effectively reported the dynamic process of the biodistribution and clearance of the nanoparticles in vivo.
IRDye800CW-labeled Fe3O4 nanoparticles provide an effective probe for cell-labeling and in vivo imaging.
Gene therapy offers the potential of mediating disease through modification of specific cellular functions of target cells. However, effective transport of nucleic acids to target cells with minimal side effects remains a challenge despite the use of unique viral and non-viral delivery approaches. Here we present a non-viral nanoparticle gene carrier that demonstrates effective gene delivery and transfection both in vitro and in vivo. The nanoparticle system (NP-CP-PEI) is made of a superparamagnetic iron oxide nanoparticle (NP), which enables magnetic resonance imaging, coated with a novel copolymer (CP-PEI) comprised of short chain polyethylenimine (PEI) and poly(ethylene glycol) (PEG) grafted to the natural polysaccharide, chitosan (CP), which allows efficient loading and protection of the nucleic acids. The function of each component material in this nanoparticle system is illustrated by comparative studies of three nanoparticle systems of different surface chemistries, through material property characterization, DNA loading and transfection analyses, and toxicity assessment. Significantly, NP-CP-PEI demonstrates an innocuous toxic profile and a high level of expression of the delivered plasmid DNA in a C6 xenograft mouse model, making it a potential candidate for safe in vivo delivery of DNA for gene therapy.
biomaterials; superparamagnetic nanoparticles; DNA; drug delivery; gene therapy
A synthetically diverse linker molecule consisting of both a terminal epoxide and a terminal amine has been synthesized and shown to have the desired reactivity. Proof of principle experimentation showed that the prepared linker molecule possessed the ability to be reactive towards dextran coated iron nanoparticles, essentially converting the surface alcohols to amines with an efficiency on average of 50 linkers per nanoparticle. Once the surface of the nanoparticles had been functionalized, the iron nanoparticles were subsequently functionalized with both folic acid and fluorescein isothiocyanate, with an average efficiency of 20 and 3 molecules per nanoparticle, respectively. The labeled nanoparticles were then incubated with both folate receptor positive and negative cell lines, which showed a preferential accumulation of the particles in the receptor positive cell line. In addition to the fluorescence based assays, accumulation of the nanoparticles was demonstrated using T2-weighted MRI imaging, which showed that the iron core of the nanoparticle was present within the desired cell line. Overall, this linker has shown the ability to functionalize the surface of nanoparticles and can theoretically be used to label a wide variety of other targeting agents or imaging agents for in vivo therapies or diagnostics.
Background & Aims
Identification of a ligand/receptor system that enables functionalized nanoparticles to efficiently target pancreatic cancer holds great promise for the development of novel approaches for the detection and treatment of pancreatic cancer. Urokinase plasminogen activator receptor (uPAR), a cellular receptor that is highly expressed in pancreatic cancer and tumor stromal cells, is an excellent surface molecule for receptor-targeted imaging of pancreatic cancer using multifunctional nanoparticles.
The uPAR-targeted dual-modality molecular imaging nanoparticle probe is designed and prepared by conjugating a near-infrared dye-labeled amino-terminal fragment of the receptor binding domain of urokinase plasminogen activator to the surface of functionalized magnetic iron oxide nanoparticles.
We have shown that the systemic delivery of uPAR-targeted nanoparticles leads to their selective accumulation within tumors of orthotopically xenografted human pancreatic cancer in nude mice. The uPAR-targeted nanoparticle probe binds to and is subsequently internalized by uPAR-expressing tumor cells and tumor-associated stromal cells, which facilitates the intratumoral distribution of the nanoparticles and increases the amount and retention of the nanoparticles in a tumor mass. Imaging properties of the nanoparticles enable in vivo optical and magnetic resonance imaging of uPAR-elevated pancreatic cancer lesions.
Targeting uPAR using biodegradable multifunctional nanoparticles allows for the selective delivery of the nanoparticles into primary and metastatic pancreatic cancer lesions. This novel receptor-targeted nanoparticle is a potential molecular imaging agent for the detection of pancreatic cancer.
To explore the capacity of human CD14+CD16++ and CD14++CD16- monocytes to phagocyte iron-oxide nanoparticles in vitro.
Human monocytes were labeled with four different magnetic nanoparticle preparations (Ferumoxides, SHU 555C, CLIO-680, MION-48) exhibiting distinct properties and cellular uptake was quantitatively assessed by flow cytometry, fluorescence microscopy, atomic absorption spectrometry and Magnetic Resonance Imaging (MRI). Additionally we determined whether cellular uptake of the nanoparticles resulted in phenotypic changes of cell surface markers.
Cellular uptake differed between the four nanoparticle preparations. However for each nanoparticle tested, CD14++CD16- monocytes displayed a significantly higher uptake compared to CD14+CD16++ monocytes, this resulted in significantly lower T1 and T2 relaxation times of these cells. The uptake of iron-oxide nanoparticles further resulted in a remarkable shift of expression of cell surface proteins indicating that the labeling procedure affects the phenotype of CD14+CD16++ and CD14++CD16- monocytes differently.
Human monocyte subsets internalize different magnetic nanoparticle preparations differently, resulting in variable loading capacities, imaging phenotypes and likely biological properties.
To develop antibody- and fluorescence-labeled superparamagnetic iron oxide nanoparticle (SPIO) “nanotheranostics” for magnetic resonance imaging (MRI) and fluorescence imaging of cancer cells and pH-dependent intracellular drug release.
SPIO nanoparticles (10 nm) were coated with amphiphilic polymers and PEGylated. The antibody HuCC49ΔCH2 and fluorescent dye 5-FAM were conjugated to the PEG of IONPs. Anticancer drugs doxorubicin (Dox), and azido-doxorubicin (Adox), MI-219, 17-DMAG containing primary amine, azide, secondary amine, and tertiary amine, respectively, were encapsulated into IONPs. The encapsulation efficiency and drug release at various pHs were determined using an LC-MS/MS. The cancer targeting and imaging were monitored using MRI and fluorescent microscopy in colon cancer cell line (LS174T). The pH-dependent drug release, intracellular distribution, and cytotoxicity were evaluated using microscopy and MTS assay.
The pegylation of SPIO and conjugation with antibody and 5-FAM increased SPIO size from 18 nm to 44 nm. Fluorescent imaging, magnetic resonance imaging (MRI) and Prussian blue staining demonstrated that HuCC49ΔCH2-SPIO increased cancer cell targeting. HuCC49ΔCH2-SPIO “nanotheranostics” decreased the T2 values in MRI of LS174T cells from 117.3±1.8 ms to 55.5±2.6 ms. The loading capacities of Dox, Adox, MI-219, and 17-DMAG were 3.16 ± 0.77%, 6.04± 0.61%, 2.22± 0.42%, and 0.09±0.07%, respectively. Dox, MI-219 and 17-DMAG showed pH-dependent release while Adox didn’t. Fluorescent imaging demonstrated the accumulation of HuCC49ΔCH2-SPIO “nanotheranostics” in endosomes/lysosomes. The encapsulated Dox was released in acidic lysosomes and diffused into cytosol and nuclei. In contrary, the encapsulated Adox only showed limited release in endosomes/lysosomes. HuCC49ΔCH2-SPIO “nanotheranostics” targetedly delivered more Dox to LS174T cells than nonspecific IgG-SPIO and resulted in a lower IC50 (1.44 μM v.s. 0.44 μM).
The developed HuCC49ΔCH2-SPIO “nanotheranostics” provides an integrated platform for cancer cell imaging, targeted anticancer drug delivery and pH-dependently drug release.
iron oxide nanoparticle (SPIO); MRI; fluorescent imaging; targeted drug delivery; nanotheranostics; doxorubicin; intracellular drug release
Magnetic resonance imaging (MRI) is widely used in modern clinical medicine as a diagnostic tool, and provides noninvasive and three-dimensional visualization of biological phenomena in living organisms with high spatial and temporal resolution. Therefore, considerable attention has been paid to magnetic nanoparticles as MRI contrast agents with efficient targeting ability and cellular internalization ability, which make it possible to offer higher contrast and information-rich images for detection of disease.
LTVSPWY peptide-modified PEGylated chitosan (LTVSPWY-PEG-CS) was synthesized by chemical reaction, and the chemical structure was confirmed by 1H-NMR. LTVSPWY-PEG-CS-modified magnetic nanoparticles were prepared successfully using the solvent diffusion method. Their particle size, size distribution, and zeta potential were measured by dynamic light scattering and electrophoretic mobility, and their surface morphology was investigated by transmission electron microscopy. To investigate their selective targeting ability, the cellular uptake of the LTVSPWY-PEG-CS-modified magnetic nanoparticles was observed in a cocultured system of SKOV-3 cells which overexpress HER2 and A549 cells which are HER2-negative. The in vitro cytotoxicity of these nanoparticles in SKOV-3 and A549 cells was measured using the MTT method. The SKOV-3-bearing nude mouse model was used to investigate the tumor targeting ability of the magnetic nanoparticles in vivo.
The average diameter and zeta potential of the LTVSPWY-PEG-CS-modified magnetic nanoparticles was 267.3 ± 23.4 nm and 30.5 ± 7.0 mV, respectively, with a narrow size distribution and spherical morphology. In vitro cytotoxicity tests demonstrated that these magnetic nanoparticles were carriers suitable for use in cancer diagnostics with low toxicity. With modification of the LTVSPWY homing peptide, magnetic nanoparticles could be selectively taken up by SKOV-3 cells overexpressing HER2 when cocultured with HER2-negative A549 cells. In vivo biodistribution results suggest that treatment with LTVSPWY-PEG-CS-modified magnetic nanoparticles/DiR enabled tumors to be identified and diagnosed more rapidly and efficiently in vivo.
LTVSPWY-PEG-CS-modified magnetic nanoparticles are a promising contrast agent for early detection of tumors overexpressing HER2 and further diagnostic application.
LTVSPWY peptide; HER2; poly(ethylene glycol); chitosan; magnetic nanoparticles; tumor targeting
Fluorescent nanoparticles with multiple emission wavelengths by a single wavelength excitation are needed in multiplex bioanalysis and molecular imaging. We have prepared silica nanoparticles encapsulated with three organic dyes using a modified Stöber synthesis method. By varying the doping ratio of the three tandem dyes, fluorescence resonance energy transfer (FRET)-mediated emission signatures can be tuned to have the nanoparticles to exhibit multiple colors under one single wavelength excitation. These nanoparticles are intensely fluorescent, highly photostable, uniform in size and biocompatible. The acceptor emission of the FRET nanoparticles has generated a large Stokes shift which implicates broad applications in biological labeling and imaging. Molecular recognition moieties, such as biotin, can be covalently attached to the nanoparticle surface to allow for specific binding to target molecules. These multicolor FRET silica nanoparticles can be used as barcoding tags for multiplexed signaling. By using these NPs, one can envision a dynamic, multicolor, colocalization methodology to follow proteins, nucleic acids, molecular machines and assemblies within living systems.
Single-molecule fluorescence methods offer the promise of ultrasensitive detection of biomolecules, but the passive immobilization methods commonly employed require analyte concentrations in the picomolar range. Here, we demonstrate that superparamagnetic Fe3O4 nanoparticles (NPs) can be used with an external magnetic field as a simple strategy to enhance the immobilization efficiency and thereby decrease the detection limit. Inorganic nanoparticles functionalized with streptavidin were bound to biotinylated single-stranded DNA oligonucleotides, which were in turn annealed to complementary oligonucleotides labeled with a Cy3 fluorescence dye. Using an external magnetic field, the superparamagnetic nanoparticles were localized to a specific region within the flow chamber surface. From the single-molecule fluorescence time traces, single-step photobleaching indicated that the surface-immobilized NPs were primarily bound with a single Cy3-labeled oligonucleotide. This strategy gave a concentration detection limit for the Cy3-labeled oligonucleotide of 100 aM, 3,000-fold lower than that from an analogous strategy with passive immobilization. With a sample volume of 25 μL, this method achieved a mole detection limit of approximately 2.5 zeptomoles (~1500 molecules). Together, the results support that idea that single-molecule fluorescence methods could be used for biological applications, such as detection and measurements of nucleic acids from biological or clinical samples without PCR amplification.
Magnetic nanoparticles; single-molecule total internal reflection fluorescence microscopy; zeptomole detection limit; directed immobilization
We report the synthesis and in vivo characterization of an 18F modified trimodal nanoparticle (18F-CLIO). This particle consists of cross-linked dextran held together in core–shell formation by a superparamagnetic iron oxide core and functionalized with the radionuclide 18F in high yield via “click” chemistry. The particle can be detected with positron emission tomography, fluorescence molecular tomography, and magnetic resonance imaging. The presence of 18F dramatically lowers the detection threshold of the nanoparticles, while the facile conjugation chemistry provides a simple platform for rapid and efficient nanoparticle labeling.
We demonstrate that novel oligonucleotide-modified gold nanoparticle probes hybridized to fluorophore-labeled complements can be used as both transfection agents and cellular “nano-flares” for detecting mRNA in living cells. Nano-flares take advantage of the highly efficient fluorescence quenching properties of gold, cellular uptake of oligonucleotide nanoparticle conjugates without the use of transfection agents, and the enzymatic stability of such conjugates, thus overcoming many of the challenges to creating sensitive and effective intracellular probes. Nano-flares exhibit high signaling, have low background fluorescence, and are sensitive to changes in the number of RNA transcripts present in cells.
Aptamer-conjugated nanoparticles (ACNPs) have been used for a variety of applications, particularly dual nanoparticles for magnetic extraction and fluorescent labeling. In this type of assay, silica-coated magnetic and fluorophore-doped silica nanoparticles are conjugated to highly selective aptamers to detect and extract targeted cells in a variety of matrices. However, considerable improvements are required in order to increase the selectivity and sensitivity of this two-particle assay to be useful in a clinical setting. To accomplish this, several parameters were investigated, including nanoparticle size, conjugation chemistry, use of multiple aptamer sequences on the nanoparticles, and use of multiple nanoparticles with different aptamer sequences. After identifying the best-performing elements, the improvements made to this assay’s conditional parameters were combined to illustrate the overall enhanced sensitivity and selectivity of the two particle assay using an innovative multiple aptamer approach, signifying a critical feature in the advancement of this technique.
Ribonucleic acid interference (RNAi) is a powerful molecular tool that has potential to revolutionize the treatment of cancer. One major challenge of applying this technology for clinical application is the lack of site-specific carriers that can effectively deliver short interfering RNA (siRNA) to cancer cells. Here we report the development and assessment of a cancer-cell specific magnetic nanovector construct for efficient siRNA delivery and non-invasive monitoring through magnetic resonance imaging (MRI). The base of the nanovector construct is comprised of a superparamagnetic iron oxide nanoparticle core coated with polyethylene glycol (PEG)-grafted chitosan, and polyethylenimine (PEI). The construct was then further functionalized with siRNA and a tumor-targeting peptide, chlorotoxin (CTX), to improve tumor specificity and potency. Flow cytometry, quantitative RT-PCR, and fluorescence microscopy analyses confirmed receptor-mediated cellular internalization of nanovectors and enhanced gene knockdown through targeted siRNA delivery. The ability of this nanovector construct to generate specific contrast enhancement of brain tumor cells was demonstrated through MR imaging. These findings suggest that this CTX enabled nanoparticle carrier may be well suited for delivery of RNAi therapeutics to cancer cells.
Nanoparticle; Gene Therapy; Cancer; Bioconjugation; MRI; siRNA
Breast cancer remains one of the most prevalent and lethal malignancies in women. The inability to diagnose small volume metastases early has limited effective treatment of stage 4 breast cancer. Here we report the rational development and use of a multifunctional superparamagnetic iron oxide nanoparticle (SPION) for targeting metastatic breast cancer in a transgenic mouse model and imaging with magnetic resonance (MR). SPIONs coated with a copolymer of chitosan and polyethylene glycol (PEG) were labeled with a fluorescent dye for optical detection and conjugated with a monoclonal antibody against the neu receptor (NP-neu). SPIONs labeled with mouse IgG were used as a non-targeting control (NP-IgG). These SPIONs had desirable physiochemical properties for in vivo applications such as near neutral zeta potential and hydrodynamic size around 40 nm, and were highly stable in serum containing medium. Only NP-neu showed high uptake in neu expressing mouse mammary carcinoma (MMC) cells which was reversed by competing free neu antibody, indicating their specificity to the neu antigen. In vivo, NP-neu was able to tag primary breast tumors and significantly, only NP-neu bound to spontaneous liver, lung, and bone marrow metastases in a transgenic mouse model of metastatic breast cancer, highlighting the necessity of targeting for delivery to metastatic disease. The SPIONs provided significant contrast enhancement in MR images of primary breast tumors; thus, they have the potential for MRI detection of micrometastases, and provide an excellent platform for further development of an efficient metastatic breast cancer therapy.
iron oxide; nanoparticle; genetically engineered mouse model; HER2/neu; theranostics; breast cancer; metastases
Tracking cells after therapeutic transplantation is imperative for evaluation of implanted cell fate and function. In this study, ultrasmall superparamagnetic iron oxide nanoparticles (USPIO NPs) were surface functionalized with water-soluble chitosan, a cationic polysaccharide that mediates enhanced endocytic uptake, endosomal escape into the cytosol, and subsequent long-term retention of nanoparticles. NP surface and chitosan were independently fluorescently labeled. Our NPs enable NP trafficking studies and determination of fate beyond uptake by fluorescence microscopy as well as tracking of labeled cells as localized regions of hypointensity in T2*-weighted magnetic resonance imaging (MRI) images. Adult rat neural stem cells (NSCs) were labeled with NPs, and assessment of NSC proliferation rates and differentiation potential revealed no significant differences between labeled and unlabeled NSCs. Significantly enhanced uptake of chitosan NPs in comparison to native NPs was confirmed by transmission electron microscopy, nuclear magnetic resonance (NMR) spectroscopy and in vitro cellular MRI at 11.7 Tesla. While only negligible fractions of native NPs enter cells, chitosan NPs appear within membranous vesicles within 2 hours of exposure. Additionally, chitosan-functionalized NPs escaped from membrane-bound vesicles within days, circumventing NP endo-lysosomal trafficking and exocytosis and hence enabling long-term tracking of labeled cells. Finally, our labeling strategy does not contain any NSC-specific reagents. To demonstrate general applicability across a variety of primary and immortalized cell types, embryonic mouse NSCs, mouse embryonic stem cells, HEK 293 kidney cells, and HeLa cervical cancer cells were additionally exposed to chitosan-USPIO NPs and exhibited similarly efficient loading as verified by NMR relaxometry. Our efficient and versatile labeling technology can support cell tracking with close to single cell resolution by MRI in vitro, for example, in complex tissue models not optically accessible by confocal or multi-photon fluorescence microscopy, and potentially in vivo, for example, in animal models of human disease or injury.
nanoparticle; iron oxide; chitosan; neural stem cell; cell tracking
Chitosan (CS) nanoparticles have been developed as a versatile drug delivery system to transport drugs, genes, proteins, and peptides into target sites. Demands on fluorescent nanoparticles have increased recently due to various applications in medical and stem-cell-based researches. In this study, fluorescent CS nanoparticles were prepared by a mild method, namely, complex coacervation. Entrapment efficiency of sulforhodamine (SR101) loaded into CS nanoparticles was investigated to evaluate their capacity in incorporating fluorescent molecule. Particle size of produced fluorescent nanoparticles was in the range of 600–700 nm, and their particle size was highly dependent on the CS molecular weight as well as concentration. A high entrapment efficiency of SR101 into CS nanoparticles could also be obtained when it was dissolved in methanol. In conclusion, highly loaded fluorescent CS nanoparticles could be easily prepared using complex coacervation method and therefore can be applied in various medical researches.
Multifunctional nanoparticles as theranostic tools hold great potential for its unique and efficient way to visualize the process of disease treatment. However, the toxicity of conventional fluorescent labels and difficulty of functionalization limit their widespread use. Recently, a number of amino-rich polymers have demonstrated high luminescent fluorescence but rarely showed potential for in vivo imaging due to their blue fluorescence. Here, a general route has been found to construct polymer-based multifunctional nanoparticles for combined imaging and drug delivering. The weak fluorescent polyethyleneimine (PEI) has been conjugated with hydrophobic polylactide as the amphiphilic PEI for construction of nanoparticles which showed bright and multicolor fluorescence with high drug loading capacity. The paclitaxel-loaded nanoparticles showed significant therapy effect in contrast to the free paclitaxel. Meanwhile, fluorescence imaging of the nanoparticles showed accumulation around tumor. These results demonstrate a new type of polymer-based multifunctional nanoparticles for imaging-guided drug delivery.