In vivo safety evaluation of polyarginine coated magnetic nanovectors.

Safety and efficacy are of critical importance to any nanomaterial-based diagnostic and therapy. The innocuity and functionality of a nanomaterial in vivo is largely dependent on the physicochemical properties of the material, particularly its surface coating. Here, we evaluated the influence of polycationic coating on the efficacy, clearance organ uptake, and safety of magnetic nanovectors designed for siRNA delivery. Polyethylene glycol (PEG) coated superparamagnetic iron oxide nanoparticles (NPs) of 12 nm in core diameter were modified with a polycationic coating of either poly-l-arginine (pArg) or polyethylenimine (PEI) and further covalently functionalized with siRNA oligonucleotides. The produced NP-pArg-siRNA and NP-PEI-siRNA nanovectors were similar in hydrodynamic size (21 and 22 nm, respectively) but significantly differed in zeta potentials (+2.1 mV and +29.8 mV, respectively). Fluorescence quantification assays revealed that the NP-pArg-siRNA nanovector was 3-fold more potent than NP-PEI-siRNA in delivering siRNA and 1.8-fold more effective in gene silencing when tested in rat C6 glioblastoma cells. In vivo, both nanovector formulations were similarly taken up by the spleen and liver as determined by histopathological and hemopathological assays. However, PEI coated nanovectors elicited severe hemoincompatibility and damage to the liver and spleen, while pArg coated nanovectors were found to be safe and tolerable. Combined, our findings suggest that polycationic coatings of pArg were more effective and safer than commonly used PEI coatings for preparation of nanovectors. The NP-pArg-siRNA nanovector formulation developed here shows great potential for in vivo based biomedical applications.

Uniaxially-aligned PVDF nanofibers as a sensor and transmitter for biotelemetry.

Biotelemetry has become an important part of medical research for patient care by remotely monitoring continuing biological processes and physiological functions. However, current biotelemetry systems are complex requiring multiple electronic components to function: a battery, a sensor, and a transmitter, and a receiver. Another paramount concern of biotelemetry is the coupling of its in vivo portion to external supporting equipment. Here we report a novel biotelemetry device made primarily of a coiled bundle of uniaxially-aligned biocompatible polyvinylidene fluoride (PVDF) nanofibers of ∼200 nm in diameter and with piezoelectric properties that can serve concurrently as a power source, sensor, and transmitter. We tested this device on a cantilever beam that was periodically deflected at its free end. Without a power supply the coil of a nanofiber bundle is shown to generate and transmit an electrical signal wirelessly in response to the beam deflection which was received by an external receiver. The coil of a nanofiber bundle was encapsulated in a thin biocompatible polymer shell for device integrity and moisture isolation. Our results suggest that the device can potentially serve as a mechanical sensor and biotelemeter for various in vitro and in vivo biomedical applications.

pH-Sensitive siRNA nanovector for targeted gene silencing and cytotoxic effect in cancer cells.

A small interfering RNA (siRNA) nanovector with dual targeting specificity and dual therapeutic effect is developed for targeted cancer imaging and therapy. The nanovector is composed of an iron oxide magnetic nanoparticle core coated with three different functional molecules: polyethyleneimine (PEI), siRNA, and chlorotoxin (CTX). The primary amine group of PEI is blocked with citraconic anhydride that is removable at acidic conditions, not only to increase its biocompatibility at physiological conditions but also to elicit a pH-sensitive cytotoxic effect in the acidic tumor microenvironment. The PEI is covalently immobilized on the nanovector via a disulfide linkage that is cleavable after cellular internalization of the nanovector. CTX as a tumor-specific targeting ligand and siRNA as a therapeutic payload are conjugated on the nanovector via a flexible and hydrophilic PEG linker for targeted gene silencing in cancer cells. With a size of ∼60 nm, the nanovector exhibits long-term stability and good magnetic property for magnetic resonance imaging. The multifunctional nanovector exhibits both significant cytotoxic and gene silencing effects at acidic pH conditions for C6 glioma cells, but not at physiological pH conditions. Our results suggest that this nanovector system could be safely used as a potential therapeutic agent for targeted treatment of glioma as well as other cancers.

Desktop-stereolithography 3D printing of a radially oriented extracellular matrix/mesenchymal stem cell exosome bioink for osteochondral defect regeneration.

Mitochondrial dysfunction and oxidative stress damage are hallmarks of osteoarthritis (OA). Mesenchymal stem cell (MSC)-derived exosomes are important in intercellular mitochondria communication. However, the use of MSC exosomes for regulating mitochondrial function in OA has not been reported. This study aimed to explore the therapeutic effect of MSC exosomes in a three dimensional (3D) printed scaffold for early OA therapeutics. Methods: We first examined the mitochondria-related proteins in normal and OA human cartilage samples and investigated whether MSC exosomes could enhance mitochondrial biogenesis in vitro. We subsequently designed a bio-scaffold for MSC exosomes delivery and fabricated a 3D printed cartilage extracellular matrix (ECM)/gelatin methacrylate (GelMA)/exosome scaffold with radially oriented channels using desktop-stereolithography technology. Finally, the osteochondral defect repair capacity of the 3D printed scaffold was assessed using a rabbit model. Results: The ECM/GelMA/exosome scaffold effectively restored chondrocyte mitochondrial dysfunction, enhanced chondrocyte migration, and polarized the synovial macrophage response toward an M2 phenotype. The 3D printed scaffold significantly facilitated the cartilage regeneration in the animal model. Conclusion: This study demonstrated that the 3D printed, radially oriented ECM/GelMA/exosome scaffold could be a promising strategy for early OA treatment.

In vivo MRI detection of gliomas by chlorotoxin-conjugated superparamagnetic nanoprobes.

Converging advances in the development of nanoparticle-based imaging probes and improved understanding of the molecular biology of brain tumors offer the potential to provide physicians with new tools for the diagnosis and treatment of these deadly diseases. However, the effectiveness of promising nanoparticle technologies is currently limited by insufficient accumulation of these contrast agents within tumors. Here a biocompatible nanoprobe composed of a poly(ethylene glycol) (PEG) coated iron oxide nanoparticle that is capable of specifically targeting glioma tumors via the surface-bound targeting peptide, chlorotoxin (CTX), is presented. The preferential accumulation of the nanoprobe within gliomas and subsequent magnetic resonance imaging (MRI) contrast enhancement are demonstrated in vitro in 9L cells and in vivo in tumors of a xenograft mouse model. TEM imaging reveals that the nanoprobes are internalized into the cytoplasm of 9L cells and histological analysis of selected tissues indicates that there are no acute toxic effects of these nanoprobes. High targeting specificity and benign biological response establish this nanoprobe as a potential platform to aid in the diagnosis and treatment of gliomas and other tumors of neuroectodermal origin.

Methotrexate-immobilized poly(ethylene glycol) magnetic nanoparticles for MR imaging and drug delivery.

We report the development of a biostable methotrexate-immobilized iron oxide nanoparticle drug carrier that may potentially be used for real-time monitoring of drug delivery through magnetic resonance imaging. Methotrexate (MTX) was immobilized on the nanoparticle surface via a poly(ethylene glycol) self-assembled monolayer (PEG SAM). The cytotoxicity of the nanoparticle-drug conjugate (NP-PEG-MTX) to target cells was studied with 9L glioma cells. Cellular uptake experiments showed that the uptake of NP-PEG-MTX conjugates by glioma cells was considerably higher than that of control nanoparticles. Magnetic resonance imaging in 9L cells cultured with NP-PEG-MTX of various concentrations showed significant contrast enhancement. NP-PEG-MTX demonstrated higher cytotoxicity in 9L cells to free MTX in vitro. Leucovorin, an MTX antidote, was used to rescue the cells that had been exposed to NP-PEG-MTX or free MTX, and the experiment verified the biocompatibility of NP-PEG-MTX conjugates and the MTX on NP-PEG-MTX conjugates to be the true source of the cytotoxicity to the target cells. TEM results showed that NP-PEG-MTX conjugates were internalized into the 9L cellular cytoplasm and retained its crystal structure therein for up to 144 h, as identified by electron diffraction. This prolonged particle retention may allow physicians to image tumor cells exposed to the NP-PEG-MTX conjugate over an extended therapeutic time course.

Wear-particle-induced osteoclast osteolysis: the role of particulates and mechanical strain.

Periprosthetic osteolysis involves osteoclast activation by wear particulates and their exposure to mechanical perturbation through exposure to shear forces generated by periprosthetic fluid as well as interface micromotion. This study aimed to determine the interactions between wear particulates, mechanical stimulation, and osteoclasts. In static cultures, wear particulates increased osteoclast differentiation. Addition of neutralizing antibodies to RANKL (receptor activator of nuclear factor kappa ligand) inhibited the particle-induced increase in osteoclast numbers. Cyclic 5000 microstrains were applied with the use of a custom-built device to marrow-derived cultures to assess the effect on osteoclast differentiation. Mechanical strain application alone decreased osteoclast differentiation, which was further decreased by the addition of particles despite increases in the soluble RANKL to osteoprotegerin (OPG) ratio. Mechanical strain alone induced mature osteoclast apoptosis in a dose-dependent manner. In contrast, in the mature osteoclast model, the addition of nonmetal particulates protected the osteoclasts from becoming apoptopic. Titanium (Ti) and cobalt chromium (CoCr) particles, however, induced osteoclast apoptosis, whereas polyethylene (PE) and polymethylmethacrylate (PMMA) did not. Wear particulates and mechanical stimulation interact via an eicosanoid-dependent pathway to alter osteoclast function and survival. The addition of mechanical perturbation to a particle-laden system thus appears to enhance the potential for osteolytic activity by enhancing osteoclast survival.

Carbonic anhydrase IX-directed immunoliposomes for targeted drug delivery to human lung cancer cells in vitro.

Targeted drug delivery to cancer cells by use of antibody-conjugated liposomes (immunoliposomes) has attracted considerable interest in recent years. Despite increasing efforts in developing immunoliposomes as drug carriers, the investigation of useful tumor-associated antigen targets is far from complete. Carbonic anhydrase IX (CA IX) is a cell surface antigen characterized by hypoxia-induced expression in many solid tumors. This study investigated the feasibility of CA IX-directed immunoliposomes for targeted delivery of docetaxel to human lung cancer cells in vitro. Docetaxel-loaded immunoliposomes targeting CA IX were developed with an encapsulation efficiency of 84.4±3.9% and an average particle size of 143.9±11.1 nm. Using fluorescence-based flow cytometry, the in vitro binding activity of the immunoliposomes was found to be significantly higher (by 1.65-fold) than that of the nontargeted liposomes in CA IX-positive lung cancer cells, whereas no such difference was observed between the two groups when CA IX was not expressed. Furthermore, immunoliposomal docetaxel exhibited the strongest growth inhibitory effect against CA IX-positive lung cancer cells when compared with nontargeted liposomal docetaxel or free docetaxel solution. These data suggested that CA IX-directed immunoliposomes could serve as a promising drug delivery system for targeted killing of lung cancer cells.

Targeted cell uptake of a noninternalizing antibody through conjugation to iron oxide nanoparticles in primary central nervous system lymphoma.

BACKGROUND: At present there is no standard of care for patients with primary central nervous system lymphoma (PCNSL) because of the difficulty in delivering therapeutically effective doses of drugs to the intracellular site of the target PCNSL. Here we report the use of an iron oxide nanoparticle to promote the internalization of a PCNSL targeting antibody by target cells. METHODS: Iron oxide nanoparticles coated with a copolymer of chitosan-grafted polyethylene glycol (NPs) were conjugated with an anti-CD20 single-chain variable fragment-streptavidin fusion protein (FP), and optically activated with Oregon Green 488. The ability of NP-FP to target PCNSL cells was assessed using flow cytometry and the ferrozine assay. Cell internalization of NP-FP was examined by confocal fluorescence microscopy. RESULTS: The antibody-conjugated NPs had a near-neutral zeta potential and remained stable in biological media for more than 1 week, which may minimizes nonspecific cell uptake. The diameter of the NPs was about 70 nm, which is in an optimal range for maximizing cell uptake. The selective binding of these NPs was demonstrated with binding to PCNSL cells 3- to 4-fold higher than binding to control cells. Z-stack imaging by confocal microscopy revealed the NPs were internalized by PCNSL cells. CONCLUSIONS: The high-degree specific binding and cell uptake of NP-FP in PCNSL suggests this NP formulation can be further developed to improve therapy of PCNSL.

Fabrication of magnetic nanoparticles with controllable drug loading and release through a simple assembly approach.

Nanoparticle-based cancer therapeutics promises to improve drug delivery safety and efficacy. However, fabrication of consistent theranostic nanoparticles with high and controllable drug loading remains a challenge, primarily due to the cumbersome, multi-step synthesis processes conventionally applied. Here, we present a simple and highly controllable method for assembly of theranostic nanoparticles, which may greatly reduce batch-to-batch variation. The major components of this nanoparticle system include a superparamagnetic iron oxide nanoparticle (SPION), a biodegradable and pH-sensitive poly (beta-amino ester) (PBAE) copolymer, a chemotherapeutic agent doxorubicin (DOX). Here the polymer pre-loaded with drug is directly assembled to the surface of SPIONs forming a drug loaded nanoparticle (NP-DOX). NP-DOX demonstrated a high drug loading efficiency of 679 µg DOX per mg iron, sustained stability in cell culture media up to 7 days, and a strong r(2) relaxivity of 146 mM(-1)•s(-1) for magnetic resonance imaging (MRI). The drug release analysis of NP-DOX showed fast DOX release at pH 5.5 and 6.4 (as in endosomal environment) and slow release at pH 7.4 (physiological condition), demonstrating pH-sensitive drug release kinetics. In vitro evaluation of NP-DOX efficacy using drug-resistant C6 glioma cells showed a 300% increase in cellular internalization at 24h post-treatment and 65% reduction of IC50 at 72 h post-treatment when compared to free DOX. These nanoparticles could serve as a foundation for building smart theranostic formulations for sensitive detection through MRI and effective treatment of cancer by controlled drug release.

Chelating Schiff base assisted azide-bridged Mn(II), Ni(II) and Cu(II) magnetic coordination polymers.

Four new Mn(II), Ni(II) and Cu(II) coordination polymers [Mn2(L1)(µ(1,1)-N3)2(µ(1,3)-N3)2]n (1), [Ni(L2)2(µ(1,3)-N3)]n(ClO4)n (2), [Cu(L3)(µ(1,1)-N3)(N3)]n (3) and [Cu(L4)(µ(1,1)-N3)2]n (4) (L1 = N,N'-bis(2-pyridylmethylene)ethane-1,2-diamine, L2 = N-(2-pyridylmethylene)methylamine, L3 = N-(2-pyridylmethylene)-3-pyridylamine, L4 = N-(2-pyridylmethylene)-tbutylamine) have been synthesized and characterized by single-crystal X-ray analysis and magnetic measurements. Complex 1 indicates a stoichiometry-dependent structural change (based on Mn:L1:N3 = 2:1:4 molar ratio) and consists of two-dimensional (2-D) (4,4) net layers, in which Mn(II) centers are co-bridged by single end-to-end (EE), double end-on (EO) azide and chelate-bridging L1 ligands. Complex 2 shows a single EE azide-bridged one-dimensional (1-D) Ni(II) chain. Complexes 3 and 4 indicate single EO and double EO azide-bridged 1-D Cu(II) chains, respectively. Complex 1 exhibits weak ferromagnetism due to its intra-layer spin-canting with T(c) = 20 K. Complex 2 shows an unusual intra-chain ferromagnetic coupling and spin-canting behaviour. Both complexes 3 and 4 exhibit intra-chain antiferromagnetic interactions. Magneto-structural parameters for these related complexes were also discussed.

Effect of cationic side-chains on intracellular delivery and cytotoxicity of pH sensitive polymer-doxorubicin nanocarriers.

Fine-tuning the design of polymer-doxorubicin conjugates permits optimization of an efficient nanocarrier to greatly increase intracellular uptake and cytotoxicity. Here, we report synthesis of a family of self-assembled polymer-doxorubicin nanoparticles and an evaluation of the effects of various types of side-chains on intracellular uptake and cytotoxicity of the nanocarriers for lymphoma cells. Monomers with three different cationic side-chains (CA) and pK(a)'s, i.e., a guanidinium group (Ag), an imidazole group (Im), and a tertiary amine group (Dm), were comparatively investigated. The cationic monomer, poly(ethylene glycol) (PEG), and doxorubicin (Dox) were reacted with 1,4-(butanediol) diacrylate (BUDA) to prepare a poly(ß-amino ester) (PBAE) polymer via Michael addition. All three polymer-Dox conjugates spontaneously formed nanoparticles (NP) through hydrophobic interactions between doxorubicin in aqueous solution, resulting in NP-Im/Dox, NP-Ag/Dox, and NP-Dm/Dox, with hydrodynamic sizes below 80 nm. Doxorubicin was linked to all 3 types of NPs with a hydrazone bond to assure selective release of doxorubicin only at acidic pH, as it occurs in the tumor microenvironment. Both NP-Im/Dox and NP-Ag/Dox exhibited much higher intracellular uptake by Ramos cells (Burkitt's lymphoma) than NP-Dm/Dox, suggesting that the type of side chain in the NPs determines the extent of intracellular uptake. As a result, NP-Im/Dox and NP-Ag/Dox showed cytotoxicity that was comparable to free Dox in vitro. Our findings suggest that the nature of surface cationic group on nanocarriers may profoundly influence their intracellular trafficking and resulting therapeutic efficacy. Thus, it is a crucial factor to be considered in the design of novel carriers for intracellular drug delivery.

Doxorubicin loaded iron oxide nanoparticles overcome multidrug resistance in cancer in vitro.

Multidrug resistance (MDR) is characterized by the overexpression of ATP-binding cassette (ABC) transporters that actively pump a broad class of hydrophobic chemotherapeutic drugs out of cancer cells. MDR is a major mechanism of treatment resistance in a variety of human tumors, and clinically applicable strategies to circumvent MDR remain to be characterized. Here we describe the fabrication and characterization of a drug-loaded iron oxide nanoparticle designed to circumvent MDR. Doxorubicin (DOX), an anthracycline antibiotic commonly used in cancer chemotherapy and substrate for ABC-mediated drug efflux, was covalently bound to polyethylenimine via a pH sensitive hydrazone linkage and conjugated to an iron oxide nanoparticle coated with amine terminated polyethylene glycol. Drug loading, physiochemical properties and pH lability of the DOX-hydrazone linkage were evaluated in vitro. Nanoparticle uptake, retention, and dose-dependent effects on viability were compared in wild-type and DOX-resistant ABC transporter over-expressing rat glioma C6 cells. We found that DOX release from nanoparticles was greatest at acidic pH, indicative of cleavage of the hydrazone linkage. DOX-conjugated nanoparticles were readily taken up by wild-type and drug-resistant cells. In contrast to free drug, DOX-conjugated nanoparticles persisted in drug-resistant cells, indicating that they were not subject to drug efflux. Greater retention of DOX-conjugated nanoparticles was accompanied by reduction of viability relative to cells treated with free drug. Our results suggest that DOX-conjugated nanoparticles could improve the efficacy of chemotherapy by circumventing MDR.

Chlorotoxin labeled magnetic nanovectors for targeted gene delivery to glioma.

Glioma accounts for 80% of brain tumors and currently remains one of the most lethal forms of cancers. Gene therapy could potentially improve the dismal prognosis of patients with glioma, but this treatment modality has not yet reached the bedside from the laboratory due to the lack of safe and effective gene delivery vehicles. In this study we investigate targeted gene delivery to C6 glioma cells in a xenograft mouse model using chlorotoxin (CTX) labeled nanoparticles. The developed nanovector consists of an iron oxide nanoparticle core, coated with a copolymer of chitosan, polyethylene glycol (PEG), and polyethylenimine (PEI). Green fluorescent protein (GFP) encoding DNA was bound to these nanoparticles, and CTX was then attached using a short PEG linker. Nanoparticles without CTX were also prepared as a control. Mice bearing C6 xenograft tumors were injected intravenously with the DNA-bound nanoparticles. Nanoparticle accumulation in the tumor site was monitored using magnetic resonance imaging and analyzed by histology, and GFP gene expression was monitored through Xenogen IVIS fluorescence imaging and confocal fluorescence microscopy. Interestingly, the CTX did not affect the accumulation of nanoparticles at the tumor site but specifically enhanced their uptake into cancer cells as evidenced by higher gene expression. These results indicate that this targeted gene delivery system may potentially improve treatment outcome of gene therapy for glioma and other deadly cancers.

PEG-mediated synthesis of highly dispersive multifunctional superparamagnetic nanoparticles: their physicochemical properties and function in vivo.

Multifunctional superparamagnetic nanoparticles have been developed for a wide range of applications in nanomedicine, such as serving as tumor-targeted drug carriers and molecular imaging agents. To function in vivo, the development of these novel materials must overcome several challenging requirements including biocompatibility, stability in physiological solutions, nontoxicity, and the ability to traverse biological barriers. Here we report a PEG-mediated synthesis process to produce well-dispersed, ultrafine, and highly stable iron oxide nanoparticles for in vivo applications. Utilizing a biocompatible PEG coating bearing amine functional groups, the produced nanoparticles serve as an effective platform with the ability to incorporate a variety of targeting, therapeutic, or imaging ligands. In this study, we demonstrated tumor-specific accumulation of these nanoparticles through both magnetic resonance and optical imaging after conjugation with chlorotoxin, a peptide with high affinity toward tumors of the neuroectodermal origin, and Cy5.5, a near-infrared fluorescent dye. Furthermore, we performed preliminary biodistribution and toxicity assessments of these nanoparticles in wild-type mice through histological analysis of clearance organs and hematology assay, and the results demonstrated the relative biocompatibility of these nanoparticles.

Specific targeting of brain tumors with an optical/magnetic resonance imaging nanoprobe across the blood-brain barrier.

Nanoparticle-based platforms have drawn considerable attention for their potential effect on oncology and other biomedical fields. However, their in vivo application is challenged by insufficient accumulation and retention within tumors due to limited specificity to the target, and an inability to traverse biological barriers. Here, we present a nanoprobe that shows an ability to cross the blood-brain barrier and specifically target brain tumors in a genetically engineered mouse model, as established through in vivo magnetic resonance and biophotonic imaging, and histologic and biodistribution analyses. The nanoprobe is comprised of an iron oxide nanoparticle coated with biocompatible polyethylene glycol-grafted chitosan copolymer, to which a tumor-targeting agent, chlorotoxin, and a near-IR fluorophore are conjugated. The nanoprobe shows an innocuous toxicity profile and sustained retention in tumors. With the versatile affinity of the targeting ligand and the flexible conjugation chemistry for alternative diagnostic and therapeutic agents, this nanoparticle platform can be potentially used for the diagnosis and treatment of a variety of tumor types.

Synthesis of Cysteine-Capped Zn(x)Cd(1)(-)(x)Se alloyed quantum dots emitting in the blue-green spectral range.

Alloyed ZnxCd1-xSe quantum dots (QDs) have been successfully prepared at low temperatures by reacting a mixture of Cd(ClO4)2 and Zn(ClO4)2 with NaHSe using cysteine as a surface-stabilizing agent. The photoluminescence (PL) spectra of the alloyed QDs are determined on the basis of the Zn2+/Cd2+ molar ratio, reaction pH, intrinsic Zn2+and Cd2+ reactivities toward NaHSe, concentration of NaHSe, and the kind of thiols. A systematic blue shift in emission wavelength of the alloyed QDs was found with the increase in the Zn mole fraction. This result provides clear evidence of the formation of ZnxCd1-xSe QDs by the simultaneous reaction of Zn2+ and Cd2+ with NaHSe, rather than the formation of separate CdSe and ZnSe nanocrystals or core-shell structure CdSe/ZnSe nanocrystals. The size and inner structure of these QDs are also corroborated by using high-resolution transmission electron microscopy and X-ray powder diffraction. To further understand the formation mechanism, the growth kinetics of Zn0.99Cd0.01Se was studied by measuring the PL spectra at different growth intervals. The results demonstrated that, in the initial stage of growth, Zn0.99Cd0.01Se has a structure with a Cd-rich core and a Zn-rich shell. The post-preparative irradiation of these QDs improved their PL properties, resulting in stronger emission.