In vivo MRI assessment of bioactive magnetic iron oxide/human serum albumin nanoparticle delivery into the posterior segment of the eye in a rat model of retinal degeneration.

BACKGROUND: Retinal degeneration diseases affect millions of patients worldwide and lead to incurable vision loss. These diseases are caused by pathologies in the retina and underlying choroid, located in the back of the eye. One of the major challenges in the development of treatments for these blinding diseases is the safe and efficient delivery of therapeutics into the back of the eye. Previous studies demonstrated that narrow size distribution core-shell near infra-red fluorescent iron oxide (IO) nanoparticles (NPs) coated with human serum albumin (HSA, IO/HSA NPs) increase the half-life of conjugated therapeutic factors, suggesting they may be used for sustained release of therapeutics. In the present study, the in vivo tracking by MRI and the long term safety of IO/HSA NPs delivery into the suprachoroid of a rat model of retinal degeneration were assessed. RESULTS: Twenty-five Royal College of Surgeons (RCS) pigmented rats received suprachoroidal injection of 20-nm IO/HSA NPs into the right eye. The left eye was not injected and used as control. Animals were examined by magnetic resonance imaging (MRI), electroretinogram (ERG) and histology up to 30 weeks following injection. IO/HSA NPs were detected in the back part of the rats' eyes up to 30 weeks following injection by MRI, and up to 6 weeks by histology. No significant differences in retinal structure and function were observed between injected and non-injected eyes. There was no significant difference in the weight of IO/HSA NP-injected animals compared to non-injected rats. CONCLUSIONS: MRI could track the nanoparticles in the posterior segment of the injected eyes demonstrating their long-term persistence, and highlighting the possible use of MRI for translational studies in animals and in future clinical studies. Suprachoroidal injection of IO/HSA NPs showed no sign of adverse effects on retinal structure and function in a rat model of retinal degeneration, suggesting that suprachoroidal delivery of IO/HSA NPs is safe and that these NPs may be used in future translational and clinical studies for extended release drug delivery at the back of the eye.

Near-Infrared Light Induced Phase Transition of Biodegradable Composites for On-Demand Healing and Drug Release.

Light responsive materials play an important role in many biomedical applications. Despite the great potential, commonly available systems are limited by their toxicity and lack of biodegradability. Here, an efficient light triggered system from safe, biodegradable star-poly(ethylene glycol) (star-PEG) and poly(ε-caprolactone) (PCL) with varying melting points controlled by the length of the CL segments is described. When incorporated with gold nanoshells (GNS) and exposed to near-infrared (NIR) irradiation, matrices temporarily disengage, thus allowing efficient on-demand healing and drug release. The responsiveness of this system to light, with its tailorable physical and healing properties, biocompatibility, biodegradability, and the capability to incorporate drugs and on-demand drug release are all desirable traits for numerous clinical applications.

Amphiphilic star PEG-Camptothecin conjugates for intracellular targeting.

Camptothecin (CPT) is a naturally occurring cytotoxic alkaloid having a broad spectrum of antitumor activity. Unfortunately, it has low bioavailability and encapsulation efficiency, limiting its clinical use. We report on our efforts to develop a novel drug delivery prototype composed of a short, star hydrophilic polyethylene glycol (PEG) backbone and hydrophobic CPT (PEG4-CPT). The amphiphilic bio-conjugate self-assembles in water into stable spherical nano-particles with a mean diameter of 200nm and CPT substitution percentage of 27%w/w. CPT is released in a sustained release profile without burst effect. In addition, PEG4-CPT nano-particles are able to load a co-drug, water soluble or non-water soluble doxorubicin and release them simultaneously with the free CPT. The biological evaluation of PEG4-CPT against HeLa cells showed improved cellular uptake and enhanced cytotoxicity compared to free CPT. Thus, in this approach CPT acts in two ways: As the hydrophobic segment that enables self-assembly in water and as a potent anticancer agent. This concept of combining hydrophobic drugs and short star polymers shows great potential for efficient delivery of hydrophobic chemotrophic drugs as well as for drugs with inherent stability and pharmacokinetic barriers.

Synthesis and characterization of crosslinked polyisothiouronium methylstyrene nanoparticles of narrow size distribution for antibacterial and antibiofilm applications.

BACKGROUND: Isothiouronium salts are well known in their variety of antimicrobials activities. The use of polymeric biocides, polymers with antimicrobial activities, is expected to enhance the efficacy of some existing antimicrobial agents, thus minimizing the environmental problems accompanying conventional antimicrobials. METHODS: The current manuscript describes the synthesis and characterization of crosslinked polyisothiouronium methylstyrene (PITMS) nanoparticles (NPs) of narrow size distribution by dispersion co-polymerization of the monomer isothiouronium methylstyrene with the crosslinking monomer ethylene glycol dimetacrylate. RESULTS AND DISCUSSION: The effect of total monomer, crosslinker and initiator concentrations on the size and size distribution of the formed NPs was also elucidated. The bactericidal activity of PITMS NPs of 67 ± 8 nm diameter was illustrated for 4 bacterial pathogens: Listeria innocua, Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus. In order to demonstrate the potential of these PITMS NPs as inhibitor of biofilm formation, polyethylene terephthalate (PET) films were thin-coated with the PITMS NPs. The formed PET/PITMS films reduced the viability of the biofilm of Listeria by 2 orders of magnitude, making the coatings excellent candidates for further development of non-fouling surfaces. In addition, PITMS NP coatings were found to be non-toxic in HaCaT cells. CONCLUSIONS: The high antibacterial activity and effective inhibition of bacterial adsorption indicate the potential of these nanoparticles for development of new types of antibacterial and antibiofilm additives.

Bioactive magnetic near Infra-Red fluorescent core-shell iron oxide/human serum albumin nanoparticles for controlled release of growth factors for augmentation of human mesenchymal stem cell growth and differentiation.

BACKGROUND: Iron oxide (IO) nanoparticles (NPs) of sizes less than 50 nm are considered to be non-toxic, biodegradable and superparamagnetic. We have previously described the generation of IO NPs coated with Human Serum Albumin (HSA). HSA coating onto the IO NPs enables conjugation of the IO/HSA NPs to various biomolecules including proteins. Here we describe the preparation and characterization of narrow size distribution core-shell NIR fluorescent IO/HSA magnetic NPs conjugated covalently to Fibroblast Growth Factor 2 (FGF2) for biomedical applications. We examined the biological activity of the conjugated FGF2 on human bone marrow mesenchymal stem cells (hBM-MSCs). These multipotent cells can differentiate into bone, cartilage, hepatic, endothelial and neuronal cells and are being studied in clinical trials for treatment of various diseases. FGF2 enhances the proliferation of hBM-MSCs and promotes their differentiation toward neuronal, adipogenic and osteogenic lineages in vitro. RESULTS: The NPs were characterized by transmission electron microscopy, dynamic light scattering, ultraviolet-visible spectroscopy and fluorescence spectroscopy. Covalent conjugation of the FGF2 to the IO/HSA NPs significantly stabilized this growth factor against various enzymes and inhibitors existing in serum and in tissue cultures. IO/HSA NPs conjugated to FGF2 were internalized into hBM-MSCs via endocytosis as confirmed by flow cytometry analysis and Prussian Blue staining. Conjugated FGF2 enhanced the proliferation and clonal expansion capacity of hBM-MSCs, as well as their adipogenic and osteogenic differentiation to a higher extent compared with the free growth factor. Free and conjugated FGF2 promoted the expression of neuronal marker Microtubule-Associated Protein 2 (MAP2) to a similar extent, but conjugated FGF2 was more effective than free FGF2 in promoting the expression of astrocyte marker Glial Fibrillary Acidic Protein (GFAP) in these cells. CONCLUSIONS: These results indicate that stabilization of FGF2 by conjugating the IO/HSA NPs can enhance the biological efficacy of FGF2 and its ability to promote hBM-MSC cell proliferation and trilineage differentiation. This new system may benefit future therapeutic use of hBM-MSCs.

Synthesis and characterization of bioactive conjugated near-infrared fluorescent proteinoid-poly(L-lactic acid) hollow nanoparticles for optical detection of colon cancer.

Colon cancer is one of the major causes of death in the Western world. Early detection significantly improves long-term survival for patients with colon cancer. Near-infrared (NIR) fluorescent nanoparticles are promising candidates for use as contrast agents for tumor detection. Using NIR offers several advantages for bioimaging compared with fluorescence in the visible spectrum: lower autofluorescence of biological tissues and lower absorbance and, consequently, deeper penetration into biomatrices. The present study describes the preparation of new NIR fluorescent proteinoid-poly(L-lactic acid) (PLLA) nanoparticles. For this purpose, a P(EF-PLLA) random copolymer was prepared by thermal copolymerization of L-glutamic acid (E) with L-phenylalanine (F) and PLLA. Under suitable conditions, this proteinoid-PLLA copolymer can self-assemble to nanosized hollow particles of relatively narrow size distribution. This self-assembly process was used for encapsulation of the NIR dye indocyanine green. The encapsulation process increases significantly the photostability of the dye. These NIR fluorescent nanoparticles were found to be stable and nontoxic. Leakage of the NIR dye from these nanoparticles into phosphate-buffered saline containing 4% human serum albumin was not detected. Tumor-targeting ligands such as peanut agglutinin and anticarcinoembryonic antigen antibodies were covalently conjugated to the surface of the NIR fluorescent P(EF-PLLA) nanoparticles, thereby increasing the fluorescent signal of tumors with upregulated corresponding receptors. Specific colon tumor detection by the NIR fluorescent P(EF-PLLA) nanoparticles was demonstrated in a chicken embryo model. In future work, we plan to extend this study to a mouse model, as well as to encapsulate a cancer drug such as doxorubicin within these nanoparticles for therapeutic applications.

Engineering of near infrared fluorescent proteinoid-poly(L-lactic acid) particles for in vivo colon cancer detection.

BACKGROUND: The use of near-infrared (NIR) fluorescence imaging techniques has gained great interest for early detection of cancer owing to the negligible absorption and autofluorescence of water and other intrinsic biomolecules in this region. The main aim of the present study is to synthesize and characterize novel NIR fluorescent nanoparticles based on proteinoid and PLLA for early detection of colon tumors. METHODS: The present study describes the synthesis of new proteinoid-PLLA copolymer and the preparation of NIR fluorescent nanoparticles for use in diagnostic detection of colon cancer. These fluorescent nanoparticles were prepared by a self-assembly process in the presence of the NIR dye indocyanine green (ICG), a FDA-approved NIR fluorescent dye. Anti-carcinoembryonic antigen antibody (anti-CEA), a specific tumor targeting ligand, was covalently conjugated to the P(EF-PLLA) nanoparticles through the surface carboxylate groups using the carbodiimide activation method. RESULTS AND DISCUSSION: The P(EF-PLLA) nanoparticles are stable in different conditions, no leakage of the encapsulated dye into PBS containing 4% HSA was detected. The encapsulation of the NIR fluorescent dye within the P(EF-PLLA) nanoparticles improves significantly the photostability of the dye. The fluorescent nanoparticles are non-toxic, and the biodistribution study in a mouse model showed they evacuate from the body over 24 h. Specific colon tumor detection in a chicken embryo model and a mouse model was demonstrated for anti-CEA-conjugated NIR fluorescent P(EF-PLLA) nanoparticles. CONCLUSIONS: The results of this study suggest a significant advantage of NIR fluorescence imaging using NIR fluorescent P(EF-PLLA) nanoparticles over colonoscopy. In future work we plan to broaden this study by encapsulating cancer drugs such as paclitaxel and/or doxorubicin, within these biodegradable NIR fluorescent P(EF-PLLA) nanoparticles, for both detection and therapy of colon cancer.

Synthesis and characterization of near IR fluorescent albumin nanoparticles for optical detection of colon cancer.

Near IR (NIR) fluorescent human serum albumin (HSA) nanoparticles hold great promise as contrast agents for tumor diagnosis. HSA nanoparticles are considered to be biocompatible, non-toxic and non-immunogenic. In addition, NIR fluorescence properties of these nanoparticles are important for in vivo tumor diagnostics, with low autofluorescence and relatively deep penetration of NIR irradiation due to low absorption of biomatrices. The present study describes the synthesis of new NIR fluorescent HSA nanoparticles, by entrapment of a NIR fluorescent dye within the HSA nanoparticles, which also significantly increases the photostability of the dye. Tumor-targeting ligands such as peanut agglutinin (PNA) and anti-carcinoembryonic antigen antibodies (anti-CEA) were covalently conjugated to the NIR fluorescent albumin nanoparticles, increasing the potential fluorescent signal in tumors with upregulated corresponding receptors. Specific colon tumor detection by the NIR fluorescent HSA nanoparticles was demonstrated in a chicken embryo model and a rat model. In future work we also plan to encapsulate cancer drugs such as doxorubicin within the NIR fluorescent HSA nanoparticles for both colon cancer imaging and therapy.

Design of near-infrared fluorescent bioactive conjugated functional iron oxide nanoparticles for optical detection of colon cancer.

BACKGROUND: Colon cancer is one of the major causes of death in the Western world. Early detection significantly improves long-term survival for patients with the disease. Near- infrared (NIR) fluorescent nanoparticles hold great promise as contrast agents for tumor detection. NIR offers several advantages for bioimaging compared with fluorescence in the visible spectrum, ie, lower autofluorescence of biological tissues, lower absorbance, and consequently deeper penetration into biomatrices. METHODS AND RESULTS: NIR fluorescent iron oxide nanoparticles with a narrow size distribution were prepared by nucleation, followed by controlled growth of thin iron oxide films onto cyanine NIR dye conjugated gelatin-iron oxide nuclei. For functionalization, and in order to increase the NIR fluorescence intensity, the NIR fluorescent iron oxide nanoparticles obtained were coated with human serum albumin containing cyanine NIR dye. Leakage of the NIR dye from these nanoparticles into phosphate-buffered saline solution containing 4% albumin was not detected. The work presented here is a feasibility study to test the suitability of iron oxide-human serum albumin NIR fluorescent nanoparticles for optical detection of colon cancer. It demonstrates that encapsulation of NIR fluorescent dye within these nanoparticles significantly reduces photobleaching of the dye. Tumor-targeting ligands, peanut agglutinin and anticarcinoembryonic antigen antibodies (αCEA), were covalently conjugated with the NIR fluorescent iron oxide-human serum albumin nanoparticles via a poly(ethylene glycol) spacer. Specific colon tumor detection was demonstrated in chicken embryo and mouse models for both nonconjugated and the peanut agglutinin-conjugated or αCEA-conjugated NIR fluorescent iron oxide-human serum albumin nanoparticles. CONCLUSION: Conjugation of peanut agglutinin or αCEA to the nanoparticles significantly increased the fluorescence intensity of the tagged colon tumor tissues relative to the nonconjugated nanoparticles.

Convection-enhanced delivery of methotrexate-loaded maghemite nanoparticles.

Convection-enhanced delivery (CED) is a novel approach for delivering drugs directly into brain tumors by intracranial infusion, enabling the distribution of high drug concentrations over large tissue volumes. This study was designed to present a method for binding methotrexate (MTX) to unique crystalline, highly ordered and superparamagnetic maghemite nanoparticles via human serum albumin (HSA) coating, optimized for CED treatments of gliomas. Naked nanoparticles and HSA- or polyethylene glycol (PEG)-coated nanoparticles with/without MTX were studied. In vitro results showed no toxicity and a similar cell-kill efficacy of the MTX-loaded particles via HSA coating to that of free MTX, while MTX-loaded particles via PEG coating showed low efficacy. In vivo, the PEG-coated nanoparticles provided the largest distributions in normal rat brain and long clearance times, but due to their low efficacy in vitro, were not considered optimal. The naked nanoparticles provided the smallest distributions and shortest clearance times. The HSA-coated nanoparticles (with/without MTX) provided good distributions and long clearance times (nearly 50% of the distribution volume remained in the brain 3 weeks post treatment). No MTX-related toxicity was noted. These results suggest that the formulation in which HSA was bound to our nanoparticles via a unique precipitation method, and MTX was bound covalently to the HSA, could enable efficient and stable drug loading with no apparent toxicity. The cell-kill efficacy of the bound MTX remained similar to that of free MTX, and the nanoparticles presented efficient distribution volumes and slow clearance times in vivo, suggesting that these particles are optimal for CED.