Tracking Osteoarthritis Progress through Cationic Nanoprobe-Enhanced Photoacoustic Imaging of Cartilage.

A major obstacle in osteoarthritis (OA) theranostics is the lack of a timely and accurate monitoring method. It is hypothesized that the loss of anionic glycosaminoglycans (GAGs) in articular cartilage reflects the progression of OA. Thus, this study investigated the feasibility of photoacoustic imaging (PAI) applied for monitoring the in vivo course of OA progression via GAG-targeted cationic nanoprobes. The nanoprobes were synthesized through electrostatic attraction between poly-l-Lysine and melanin (PLL-MNPs). Cartilage explants with different concentrations of GAGs incubated with PLL-MNPs to test the relationship between GAGs content and PA signal intensity. GAG activity was then evaluated in vivo in destabilization of the medial meniscus (DMM) surgically-induced mouse model. To track OA progression over time, mice were imaged consistently for 10 weeks after OA-inducing surgery. X-ray was used to verify the superiority of PAI in detecting OA. The correlation between PAI data and histologic results was also analyzed. In vitro study demonstrated the ability of PLL-MNPs in sensitively detecting different GAGs concentrations. In vivo PAI exhibited significantly lower signal intensity from OA knees compared to normal knees. More importantly, PA signal intensity showed serial reduction over the course of OA, while X-ray showed visible joint destruction until 6 weeks. A decrease in GAGs content was confirmed by histologic examinations; moreover, histologic findings were well correlated with PAI results. Therefore, using cationic nanoprobe-enhanced PAI to detect the changes in GAG contents provides sensitive and consistent visualization of OA development. This approach will further facilitate OA theranostics and clinical translation. STATEMENT OF SIGNIFICANCE: The study of in vivo monitoring osteoarthritis (OA) is of high significance to tracking the trajectory of OA development and therapeutic monitoring. Here, we developed a cartilage-targeted cationic nanoprobe, poly-l-Lysine-melanin nanoparticles (PLL-MNPs), enhancing photoacoustic imaging (PAI) to monitor the progression of OA. The in vitro study demonstrated the ability of PLL-MNPs to detect different concentrations of GAGs with high sensitivity. We found that the contents of GAGs in vivo steadily decreased from the development of OA initial-stage to the end-point of our investigation via PAI; it reflected the course of OA in living subjects with high sensitivity. These results allow for further development in various aspects of OA research. It has potential for clinical translation and has a great impact on personalized medicine.

Melanin-loaded biocompatible photosensitive nanoparticles for controlled drug release in combined photothermal-chemotherapy guided by photoacoustic/ultrasound dual-modality imaging.

Combined photothermal-chemotherapy guided by multimodal imaging is a promising strategy for cancer diagnosis and treatment. Multifunctional nanoparticles, such as those comprising organic and inorganic compounds, have been extensively investigated for combined photothermal-chemotherapy; however, their application is still limited by their potential long-term toxicity and lack of contrast properties. To solve these problems, in this study, a new type of multifunctional nanoparticle for combined photothermal-chemotherapy guided by dual-modality imaging was prepared with endogenous melanin by multistep emulsification to enhance tumor ablation. The nanoparticles were coated with poly(lactide-co-glycolic acid) (PLGA) and loaded with paclitaxel (PTX), encapsulated melanin and perfluoropentane (PFP). The materials in the nanoparticles were endogenous, ensuring high stability, biocompatibility, and biosafety. Nanoparticles irradiated with a laser, which induced their phase transformation into microbubbles, exhibited high photothermal conversion efficiency, thereby achieving photoacoustic (PA)/ultrasound (US) dual-modality imaging to determine tumor location, boundary, and size and to monitor drug distribution. Furthermore, optical droplet vaporization (ODV) of the nanoparticles could trigger the release of PTX; thus, these nanoparticles are a useful drug carrier. In vivo and in vitro experiments revealed that a strong synergistic antitumor effect was achieved by combining the photothermal properties of the nanoparticles with a chemotherapy drug. Importantly, the cavitation, thermoelastic expansion, and sonoporation caused by the phase transformation of the nanoparticles could directly damage the tumors. These processes also promoted the release, penetration and absorption of the drug, further enhancing the effect of combined photothermal-chemotherapy on tumor suppression. Therefore, the multifunctional nanoparticles prepared in this study provide a new strategy of using endogenous materials for controlled near-infrared (NIR)-responsive drug release and combined photothermal-chemotherapy guided by multimodal imaging.

Eumelanin-releasing spongy-like hydrogels for skin re-epithelialization purposes.

Melanin function in the skin has been associated with pigmentation but other properties such as electrical conductance, photoprotection, and antioxidant and antimicrobial activity have also been recognized. Nonetheless, the use of melanin in a skin wound healing context has never been considered. In this sense, eumelanin particles with a typical round and nano-sized morphology and electrical conductivity of 2.09 × 10-8 S cm-1 were extracted from the ink of Sepia officinalis. The ability of primary human keratinocytes (hKCs) to phagocyte eumelanin, which was then accumulated in cytosolic vesicles and nuclei surroundings, was demonstrated. Keratinocyte viability and maturation was not affected by eumelanin contact, but at eumelanin amounts higher than 0.1 mg l-1 cell morphology was altered and cell proliferation was inhibited. A time and eumelanin amount-dependent reduction of reactive oxygen species (ROS) released by eumelanin-containing ultraviolet (UV)-irradiated keratinocytes was observed. Eumelanin-containing gellan gum (GG) spongy-like hydrogels allowed a sustained release of eumelanin in the range of 0.1 to 5 mg l-1, which was shown in vitro to not be harmful to hKCs, and the absence of a strong host reaction after subcutaneous implantation in mice. Herein, we propose spongy-like hydrogels as sustained release matrices of S. officinalis eumelanin for predicting a beneficial role in skin wound healing through a direct effect over keratinocytes.

The Relationship of Brimonidine Concentration in Vitreous Body to the Free Concentration in Retina/Choroid Following Topical Administration in Pigmented Rabbits.

PURPOSE: Several studies showed that repeated topical administration of brimonidine tartrate ophthalmic solution reached the human vitreous concentration above 2 nM, which is the concentration necessary to activate the α2-adrenergic receptor. The purpose of this study was to elucidate the relationship of the brimonidine concentration in the vitreous body to the free concentration in the retina/choroid which is the target site of brimonidine on neuroprotective effect after topical administration. MATERIALS AND METHODS: Brimonidine concentrations in the eye tissues of pigmented rabbits were determined following single ocular administration of 0.1% brimonidine tartrate ophthalmic solution at pH 7.3. Binding affinity of brimonidine to melanin and melanin content in the retina/choroid of pigmented rabbits was also examined. The concentration of free brimonidine which did not bind to melanin in the retina/choroid was calculated using the binding parameters to melanin. RESULTS: Topically applied brimonidine rapidly distributed to intraocular tissues. The elimination rate from melanin-containing tissues such as the iris/ciliary body and retina/choroid was slower than the aqueous humor and vitreous body in pigmented rabbits. In both the anterior and posterior retina/choroid, the free brimonidine concentrations were over 100-fold lower than the total concentrations. The concentrations in the vitreous body closely matched to the free concentrations in the posterior retina/choroid. Simulated free concentrations in the posterior retina/choroid were gradually increased when 0.1% solution was instilled twice daily. CONCLUSION: The present data indicated that the brimonidine concentration in the vitreous body was comparable to the free concentration in the posterior retina/choroid. This suggests that the vitreous concentration can be a surrogate indicator of the free brimonidine concentration in the posterior retina/choroid. From the present findings, it is expected that multiple instillation of brimonidine tartrate ophthalmic solution may produce the sufficient free concentration for activation of the α2-adrenergic receptor in the retina/choroid in human.

Pharmacokinetics study of Zr-89-labeled melanin nanoparticle in iron-overload mice.

Melanin, a natural biological pigment present in many organisms, has been found to exhibit multiple functions. An important property of melanin is its ability to chelate metal ions strongly, which might be developed as an iron chelator for iron overload therapy. Herein, we prepared the ultrasmall water-soluble melanin nanoparticle (MP) and firstly evaluate the pharmacokinetics of MP in iron-overload mice to provide scientific basis for treating iron-overload. To study the circulation time and biodistribution, MP was labeled with (89)Zr, a long half-life (78.4h) positron-emitting metal which is suited for the labeling of nanoparticles and large bioactive molecule. MP was chelated with (89)Zr directly at pH5, resulting in non-decay-corrected yield of 89.6% and a radiochemical purity of more than 98%. The specific activity was at least190 MBq/µmol. The (89)Zr-MP was stable in human plasma and PBS for at least 48h. The half-life of (89)Zr-MP was about 15.70±1.74h in iron-overload mice. Biodistribution studies and MicroPET imaging showed that (89)Zr-MP mainly accumulated in liver and spleen, which are the target organ of iron-overload. The results indicate that the melanin nanoparticle is promising for further iron overload therapy.

Water-soluble dopamine-based polymers for photoacoustic imaging.

Here we present a facile synthetic method yielding a linear form of polydopamine via Kumada-coupling, which can be converted into water-soluble melanin, generating high contrast in photoacoustic imaging.

Mushroom tyrosinase inhibitory activity and major fatty acid constituents of Amazonian native flora oils

In order to treat hyperpigmentation-related problems, there has been a global trend in developing cosmetics claiming to have skin-whitening properties, which act by inhibiting melanin biosynthesis. The objective of this work was to evaluate the in vitro mushroom tyrosinase inhibitory activity of five Amazonian native flora oils, and so to verify the possibility of their incorporation into cosmetic products. In addition, the fatty acid composition of the essential oils was determined by gas chromatography-flame ionisation detection in order to determine the main components of these oils. The tyrosinase inhibitory activity of the tested oils was found to be in the following order: açaí (IA50 = 66.08 µg mL-1) > tucumã > patauá > pracaxi > castanha do Brasil. This study suggests that açaí oil has great potential in the treatment of hyperpigmentation and other related disorders, due to its considerable tyrosinase inhibitory activity.

Com o intuito de se tratar problemas dermatológicos de hiperpigmentação, há uma tendência mundial no desenvolvimento de cosméticos que possuam propriedades despigmentantes, os quais agem inibindo a biossíntese de melanina. O objetivo deste trabalho foi avaliar in vitro a atividade de inibição da tirosinase de cogumelo de cinco óleos de plantas nativas da Amazônia e, desta forma, verificar a possibilidade de sua incorporação em produtos cosméticos. Ainda, a composição de ácidos graxos dos óleos foi determinada por cromatografia gasosa com detecção por ionização de chama, no intuito de determinar os principais componentes destes óleos. A atividade de inibição da tirosinase dos óleos testados foi encontrada na seguinte ordem: açaí (IA50 = 66,08 µg mL-1) > tucumã > patauá > pracaxi > castanha do Brasil. Este estudo sugere que o óleo de açaí possui grande potencial para o tratamento da hiperpigmentação cutânea e doenças correlatas, devido à sua considerável atividade de inibição da tirosinase.

Melanin-covered nanoparticles for protection of bone marrow during radiation therapy of cancer.

PURPOSE: Protection of bone marrow against radiotoxicity during radioimmunotherapy and in some cases external beam radiation therapy such as hemi-body irradiation would permit administration of significantly higher doses to tumors, resulting in increased efficacy and safety of treatment. Melanin, a naturally occurring pigment, possesses radioprotective properties. We hypothesized that melanin, which is insoluble, could be delivered to the bone marrow by intravenously administrated melanin-covered nanoparticles (MNs) because of the human body's "self-sieving" ability, protecting it against ionizing radiation. METHODS AND MATERIALS: The synthesis of MNs was performed via enzymatic polymerization of 3,4-dihydroxyphenylalanine and/or 5-S-cysteinyl-3,4-dihydroxyphenylalanine on the surface of 20-nm plain silica nanoparticles. The biodistribution of radiolabeled MNs in mice was done at 3 and 24 h. Healthy CD-1 mice (Charles River Laboratories International, Inc., Wilmington, MA) or melanoma tumor-bearing nude mice were given MNs intravenously, 50 mg/kg of body weight, 3 h before either whole-body exposure to 125 cGy or treatment with 1 mCi of (188)Re-labeled 6D2 melanin-binding antibody. RESULTS: Polymerization of melanin precursors on the surface of silica nanoparticles resulted in formation of a 15-nm-thick melanin layer as confirmed by light scattering, transmission electron microscopy, and immunofluorescence. The biodistribution after intravenous administration showed than MN uptake in bone marrow was 0.3% and 0.2% of injected dose per gram at 3 and 24 h, respectively, whereas pre-injection with pluronic acid increased the uptake to 6% and 3% of injected dose per gram, respectively. Systemic MN administration reduced hematologic toxicity in mice treated with external radiation or radioimmunotherapy, whereas no tumor protection by MNs was observed. CONCLUSIONS: MNs or similar structures provide a novel approach to protection of bone marrow from ionizing radiation based on prevention of free radical formation by melanin.

Binding characteristics of fluoroquinolones to synthetic levodopa melanin.

To define the binding characteristics of fluoroquinolones to synthetic levodopa melanin, the binding of various drugs, including levofloxacin and ofloxacin, and positive controls (timolol and chloroquine), was investigated in-vitro. The affinity and capacity of the drug binding were calculated by Langmuir's adsorption isotherm. The affinity constant (K) and the binding capacity (r(max)) of levofloxacin were similar to those of timolol and much lower than those of chloroquine. Racemic ofloxacin and its enantiomers showed similar K and r(max), suggesting that the binding lacked stereoselectivity. The binding experiment with levofloxacin derivatives indicated that the basic nitrogen atom at position 7 of the quinolone ring, but not carboxyl group at position 3, would play a critical role in the interaction of fluoroquinolones with melanin. The melanin-drug complexes of levofloxacin and chloroquine were washed with neutral phosphate buffer, ethanol and 1 M HCl solution to explain the nature of the interaction of melanin with the drugs. Electrostatic forces mainly participate in the formation of the chloroquine-melanin complex, whereas van der Waals' and hydrophobic interactions are involved in the levofloxacin-melanin complex in addition to electrostatic forces. The interactions of various fluoroquinolones such as norfloxacin, enoxacin, sparfloxacin, ciprofloxacin and lomefloxacin with melanin were also studied. The results showed that the relative K value was: chloroquine approximately ciprofloxacin, sparfloxacin >/= lomefloxacin > timolol, levofloxacin approximately enoxacin, norfloxacin, and that the relative r(max) value was: norfloxacin, enoxacin >/= chloroquine, sparfloxacin > levofloxacin, ciprofloxacin, timolol, lomefloxacin. The fluoroquinolones vary in their affinity and capacity to bind with melanin, and ciprofloxacin and sparfloxacin showed a stronger interaction with melanin than the other fluoroquinolones studied.

Characterization of [Phe(13), Tyr(19)]-MCH analog binding activity to the MCH receptor.

Melanin concentrating hormone (MCH), a hypothalamic neuropeptide, is an important regulator of energy homeostasis in mammals. Characterization of an MCH specific receptor has been hampered by the lack of a suitable radioligand. The [Phe(13), Tyr(19)]-MCH analog has been shown by different investigators to bind specifically to cell lines of epithelial or pigment cell origin. Recently, using functional assays, the MCH receptor has been characterized as a seven transmembrane G-coupled protein initially identified as SLC-1. In the present study, we used tyrosine iodinated [Phe(13), Tyr(19)]-MCH analog, which stimulates food intake in a manner similar to that of MCH, as well as native MCH to conduct binding studies. Specific binding could not be demonstrated in intact cells of several cell lines, including A431 and B16. Specific binding associated with membranes localized to the microsomal, not the plasma membrane, fraction. Message for SLC-1 was absent in these cell lines, as assessed by Northern blot analysis. We conclude that cells previously reported to express the MCH receptor do not express SLC-1 and that both iodinated MCH and the [Phe(13), Tyr(19)]-MCH have a large component of non-specific binding. These ligands may be useful for binding studies in transfected cells with high levels of SLC-1 expression. However they do not appear to be suitable for screening for the MCH receptor as most cells demonstrate significant low affinity non-specific binding.

Phe(13),Tyr(19)-melanin-concentration hormone and the blood-brain barrier: role of protein binding.

Melanin-concentrating hormone (MCH), found both peripherally and centrally, is involved in food ingestion. Although its expression in brain is increased by fasting, it is not known whether it crosses the blood-brain barrier (BBB). Use of the sensitive method of multiple-time regression analysis has shown that almost all of the peptides and polypeptides tested cross the BBB at a rate faster than the vascular marker albumin. With this same method, however, we found that the 19-amino acid 125I-Phe13,Tyr19-MCH did not cross faster than 99mTc-albumin. Several mechanisms were excluded as possible explanations for the slow rate of influx. These included degradation, association with capillary endothelial cells, and transport from brain to blood. When Phe13,Tyr19-MCH was perfused in blood-free buffer, however, it entered the brain significantly faster than albumin. This suggested protein binding as an explanation for the slow rate of influx when the MCH was administered in blood. Protein binding was confirmed by capillary zone electrophoresis, which showed that almost all of the Phe13,Tyr19-MCH added to blood migrated with a large-molecular-weight substance. Sodium dodecyl sulfate-capillary gel electrophoresis of Phe13,Tyr19-MCH in buffer additionally showed that the MCH aggregated as a trimer, a factor not preventing its influx by blood-free perfusion. Thus, the results show that blood-borne Phe13,Tyr19-MCH does not significantly cross the BBB, probably because of its binding to serum proteins.

Effects of melanin on high- and low- linear energy transfer (LET) radiation response of human epithelial cells.

The search for effective radioprotectors is of major concern in the medical, military, environmental, and space sciences. Conventional radioprotectors are generally effective only during a single irradiation and display their radioprotective properties only at high, toxic concentrations. In addition, they reduce somatic radiation effects but are poorly efficient in protecting from hereditary stochastic radiation effects. In this respect, the pigment melanin merits attention. Experiments referring to potential melanin effects on the ionising radiation response have been carried out with different biological systems, both in vivo and in vitro. In this paper, we present results on the response to high- and low-linear energy transfer (LET) radiation of a human mammary epithelial cell line, H184B5 F5-1 M/10, supplemented by melanin. The incorporation of auto-oxidative (L-dopa) melanin was linear for concentrations from 3 to 10 micrograms/ml in the growth medium. Concentrations of up to 250 micrograms/ml did not significantly impair the cells proliferative ability. No significant protective effect of melanin on the survival of cultured cells after exposure to alpha-particles (130 keV/micron) or x-rays was observed.

Topical liposome delivery of molecules to hair follicles in mice.

The hair cycle consisting of growing and resting phases, is subject to widespread disease such as androgenic alopecia or loss of pigment which are in need of effective, targeted therapeutics. In order to develop a hair-follicle delivery system we demonstrate here that phosphatidylcholine liposomes entrapping either the fluorescent dye calcein or the pigment melanin can deliver these molecules into the hair follicle and hair shafts of mice when applied topically. Liposomal delivery of these molecules is time dependent. Negligible amounts of delivered molecules enter the dermis, epidermis or blood stream thereby demonstrating the enrichment of follicle delivery. Naked calcein and melanin are trapped in the stratum corneum and are unable to enter the follicle. The potential of the hair-follicle liposome delivery system for therapeutic use for hair disease is discussed.

Effects of estrogens on human melanocytes in vitro.

Subjects with elevated serum estrogen concentrations, such as those who are pregnant or ingesting estrogen-containing contraceptive medication, may develop increased skin pigmentation. As little information is available on the mechanism(s) underlying this relationship, the in vitro effects of estrogens on melanocytes cultured from normal human skin were examined. Physiological concentrations of 17 beta-estradiol (10(-11) to 10(-9) M) significantly increased the activity of tyrosinase in melanocytes from 15 of 23 subjects. The observed increases ranged from 1.2- to 2.4-fold. Melanin synthesis, which correlated with tyrosinase activity (r = 0.98, P < 0.001) was increased to a similar extent. Melanin extrusion was also increased by 17 beta-estradiol (10(-9) M). The estrogens, estriol (10(-9) M) and estrone (10(-9) M) stimulated tyrosinase activity and melanin extrusion to a lesser extent than 17 beta-estradiol. The analogue 17 alpha-estradiol (10(-9) M) was shown to have effects on melanocyte tyrosinase activity and melanin extrusion that were equivalent to those of 17 beta-estradiol. The pure estrogen antagonist ICI 164384 (10(-6) M) also stimulated tyrosinase activity. Cycloheximide (50 micrograms/ml) inhibited 17 beta-estradiol-induced tyrosinase stimulation (P < 0.001). These results indicate that several aspects of melanocyte function respond directly to estrogenic stimulation. The equivalent effects of the 17 alpha-analogue and a "pure" anti-estrogen suggest that the 17 beta-estradiol response may be mediated through a non-classical mechanism which is similar to that described in other tissues of neural crest origin.

Polyethylene-glycol-mediated delivery of liposome-entrapped pigments into fibroblasts: experimental pigment cells as models for mutator phenotypes.

The role of epidermal melanin pigments in the development of skin cancer remains unclear. A new technique for the specific introduction of compartmentalized melanin into nonpigmented human fibroblasts through the use of liposomes and polyethylene glycol (PEG) is presented. The delivery of liposome-encapsulated material to cells was characterized by: (a) high efficiency of delivery through PEG-mediated endocytosis at 37 degrees C; (b) intracellular acidification of liposome entrapped pH-sensitive 8-hydroxypyrene-3,6,8-trisulfonic acid after delivery; (c) similar incorporation and acidification of apolipoprotein E-associated liposomes into fibroblasts via the low-density lipoprotein-receptor pathway; and (d) discharge into the extracellular space after incorporation. Similar experiments were carried out with melanin-containing liposomes that were used to introduce compartmentalized melanin into fibroblasts, through PEG-mediated delivery. These "artificial" melanocytes had functional analogies to genuine melanocytes in that (a) in both cell types melanin compartmentalization was at a lower pH; and (b) liposome contents were later expelled in analogy to the putative biological process of melanin expulsion from the melanocyte. The modified fibroblasts provided potential "mutator" phenotypes with specific melanin pigmentation, and established a new basis for studying the role of melanin pigmentation in cancer development.