Intratympanic administration of alpha-lipoic acid-loaded pluronic F-127 nanoparticles ameliorates acute hearing loss.

We used antioxidant-containing nanoparticles (NPs) to treat acute hearing loss. Alpha-lipoic acid (ALA) served as the antioxidant; we employed Pluronic F127 to fabricate NPs. In vitro, ALA-NPs protected cells of the organ of Corti in HEI-OC1 mice, triggering nuclear translocation of NRF2 and increases in the levels of antioxidant proteins, including Nrf2, HO-1, SOD-1, and SOD-2. In vivo, the hearing of mice that received ALA-NP injections into the middle ear cavity was better preserved after induction of ototoxicity than in control animals. The cochlear Nrf2 level increased in test mice, indicating that the ALA-NPs protected hearing via the antioxidant mechanism observed in vitro. ALA-NPs effectively protected against acute hearing loss by activating the Nrf2/HO-1 pathway.

A comparative study of the effect of drug hydrophobicity on nanoparticle drug delivery in vivo using two photosensitizers.

To evaluate the effect of drug hydrophobicity on nanoparticle delivery in vivo, we conducted a comparative study using different photosensitizer-loaded poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs). Chlorin e6 (Ce6) and pheophorbide a (Pba) with similar structure but different hydrophobicity were loaded into PLGA-NPs separately. We observed release profiles and photodynamic effects in vitro from the resulting Ce6- and Pba-PLGA-NPs. After intravenous injection into SCC7 tumor-bearing mice, biodistribution and accumulation of two drugs in tumor tissue were observed by real-time fluorescence imaging. Finally, in vivo photodynamic therapy with Ce6- and Pba-PLGA-NPs provided different therapeutic results according to imaging data. The results demonstrated that drug hydrophobicity is an important factor in nanoparticle drug delivery and should be considered for efficient drug delivery in vivo.

Gelatin-chlorin e6 conjugate for in vivo photodynamic therapy.

BACKGROUND: Improving the water solubility of hydrophobic photosensitizer and increasing its accumulation in tumor tissue are essential for in vivo photodynamic therapy (PDT). Considering commercialization or clinical application in future, it will be promising to achieve these purposes by developing new agents with simple and non-toxic structure. RESULTS: We conjugated multiple chlorin e6 (Ce6) molecules to gelatin polymer, synthesizing two types of gelatin-Ce6 conjugates with different amounts of Ce6: gelatin-Ce6-2 and gelatin-Ce6-8. The resulting conjugates remained soluble in aqueous solutions for a longer time than hydrophobic Ce6. The conjugates could generate singlet oxygen and kill tumor cells upon laser irradiation. After intravenous injection into SCC-7 tumor-bearing mice, gelatin-Ce6-2 showed prolonged blood circulation and highly increased accumulation in tumor tissue as observed in real-time imaging in vivo. After laser irradiation, gelatin-Ce6-2 suppressed tumor growth completely and enabled improved PDT compared to free Ce6 and gelatin-Ce6-8. CONCLUSIONS: This work demonstrates that a simple structure based on photosensitizer and gelatin can highly improve water solubility and stability. Superior tumor tissue accumulation and increased therapeutic efficacy of gelatin-Ce6 during in vivo PDT showed its high potential for clinical application.

Chlorin e6-Loaded PEG-PCL Nanoemulsion for Photodynamic Therapy and In Vivo Drug Delivery.

We fabricated poly (ethylene glycol)-block-polycaprolactone (PEG-b-PCL) nanoemulsion for drug delivery and photodynamic therapy. PEG-b-PCL effectively stabilized the interface between water and soybean oil, and the resulting nanoemulsion was about 220.3 nm in diameter with spherical shape. For photodynamic therapy (PDT), chlorin e6 (Ce6) was loaded into the nanoemulsion as a photosensitizer (PS). These chlorin e6-loaded PEG-PCL nanoemulsions (Ce6-PCL-NEs) showed efficient cellular uptake and, upon laser irradiation, generated singlet oxygen to kill tumor cells. Particularly, Ce6-PCL-NEs showed prolonged blood circulation and about 60% increased tumor accumulation compared to free Ce6 after intravenous injection to 4T1 tumor-bearing mice. These results demonstrate the promising potential of Ce6-PCL-NEs for efficient PDT and in vivo drug delivery to tumor tissue.

Folate-modified PLGA nanoparticles for tumor-targeted delivery of pheophorbide a in vivo.

Targeted drug delivery has been an important issue for tumor therapy including photodynamic therapy (PDT). The purpose of our study is to increase the targeting efficiency of photosensitizer (PS) using folate-modified nanoparticles (NPs) to tumor site in vivo. Folate receptor is over-expressed on the surface of many human cancer cells. We prepared poly (lactic-co-glycolic acid) (PLGA) NPs containing pheophorbide a (Pba), a PS that is used in PDT and generates free radical for killing cancer cells. The surface of NPs was composed of phospholipids modified with polyethylene glycol (PEG) and folate (FA). The size of the resulting FA-PLGA-Pba NPs was about 200 nm in PBS at pH 7.4 and they were stable for long time. They showed faster cellular uptake to MKN28 human gastric cancer cell line than control PLGA-Pba NPs by high-affinity binding with folate receptors on cell surface. In MTT assay, FA-PLGA-Pba NPs also showed enhanced tumor cell killing compared to control PLGA-Pba NPs. In vivo and ex vivo imaging showed high accumulation of FA-PLGA-Pba NPs in tumor site during 24 h after intravenous injection to MKN28 tumor-bearing mice model. These results demonstrate that our FA-PLGA-Pba NPs are useful for tumor-targeted delivery of PS for cancer treatment by PDT.

Copper-Free Click Chemistry: Applications in Drug Delivery, Cell Tracking, and Tissue Engineering.

Traditionally, organic chemical reactions require organic solvents, toxic catalysts, heat, or high pressure. However, copper-free click chemistry has been shown to have favorable reaction rates and orthogonality in water, buffer solutions, and physiological conditions without toxic catalysts. Strain-promoted azide-alkyne cycloaddition and inverse electron-demand Diels-Alder reactions are representative of copper-free click chemistry. Artificial chemical reactions via click chemistry can also be used outside of the laboratory in a controllable manner on live cell surfaces, in the cytosol, and in living bodies. Consequently, copper-free click chemistry has many features that are of interest in biomedical research, and various new materials and strategies for its use have been proposed. Herein, recent remarkable trials that have used copper-free click chemistry are described, focusing on their applications in molecular imaging and therapy. The research is categorized as nanoparticles for drug delivery, imaging agents for cell tracking, and hydrogels for tissue engineering, which are rapidly advancing fields based on click chemistry. The content is based primarily on the experience with click chemistry-based biomaterials over the last 10 years.

Real time, high resolution video imaging of apoptosis in single cells with a polymeric nanoprobe.

We report a new apoptosis nanoprobe (Apo-NP) designed on the basis of a polymer nanoparticle platform. This simple one-step technique is capable of boosting fluorescence signals upon apoptosis in living cells, enabling real-time imaging of apoptosis in single cells and in vivo. The Apo-NP efficiently delivers chemically labeled, dual-quenched caspase-3-sensitive fluorogenic peptides into cells, allowing caspase-3-dependent strong fluorescence amplification to be imaged in apoptotic cells in real-time and at high resolution. The design platform of the Apo-NP is flexible and can be fine-tuned for a wide array of applications such as identification of caspase-related apoptosis in pathologies and for monitoring therapeutic efficacy of apoptotic drugs in cancer treatment.

In vivo vocal fold augmentation using an injectable polyethylene glycol hydrogel based on click chemistry.

It is important to focus on urgent needs in clinics and develop optimal materials. For successful augmentation of vocal folds, the ideal filler should be injectable through a syringe, and should stably maintain its volume for a long time without toxicity. To achieve these criteria, a click chemistry-based PEG (polyethylene glycol) hydrogel was developed and applied for vocal fold augmentation in vivo. The PEG hydrogel enables fast gelation in vivo after injection and provides long-term stability. Azide- and dibenzocyclooctyne (DBCO)-modified 4-arm PEG were cross-linked by chemical conjugation via click chemistry and yielded gelation within several minutes. After subcutaneous injection into mice and rats, the PEG hydrogel showed higher stability after 1 month compared to the traditionally used calcium hydroxyapatite-carboxymethyl cellulose (CaHA-CMC) filler. In rabbit models with vocal fold paralysis, the PEG hydrogel stably fixed the paralyzed vocal fold in 4 months and minimized the glottic gap. It was an improved therapeutic result compared to CaHA-CMC, demonstrating the potential of a click chemistry-based PEG hydrogel for vocal fold therapy.

Tumor-targeting peptide conjugated pH-responsive micelles as a potential drug carrier for cancer therapy.

Herein, we prepared tumor-targeting peptide (AP peptide; CRKRLDRN) conjugated pH-responsive polymeric micelles (pH-PMs) in cancer therapy by active and pH-responsive tumor targeting delivery systems, simultaneously. The active tumor targeting and tumoral pH-responsive polymeric micelles were prepared by mixing AP peptide conjugated PEG-poly(d,l-lactic acid) block copolymer (AP-PEG-PLA) into the pH-responsive micelles of methyl ether poly(ethylene glycol) (MPEG)-poly(beta-amino ester) (PAE) block copolymer (MPEG-PAE). These mixed amphiphilic block copolymers were self-assembled to form stable AP peptide-conjugated and pH-responsive AP-PEG-PLA/MPEG-PAE micelles (AP-pH-PMs) with an average size of 150 nm. The AP-pH-PMs containing 10 wt % of AP-PEG-PLA showed a sharp pH-dependent micellization/demicellization transition at the tumoral acid pH. Also, they presented the pH-dependent drug release profile at the acidic pH of 6.4. The fluorescence dye, TRITC, encapsulated AP-pH-PMs (TRITC-AP-pH-PMs) presented the higher tumor-specific targeting ability in vitro cancer cell culture system and in vivo tumor-bearing mice, compared to control pH-responsive micelles of MPEG-PAE. For the cancer therapy, the anticancer drug, doxorubicin (DOX), was efficiently encapsulated into the AP-pH-PMs (DOX-AP-pH-PMs) with a higher loading efficiency. DOX-AP-pH-PMs efficiently deliver anticancer drugs in MDA-MB231 human breast tumor-bearing mice, resulted in excellent anticancer therapeutic efficacy, compared to free DOX and DOX encapsulated MEG-PAE micelles, indicating the excellent tumor targeting ability of AP-pH-PMs. Therefore, these tumor-targeting peptide-conjugated and pH-responsive polymeric micelles have great potential application in cancer therapy.

Selection and identification of a novel bone-targeting peptide for biomedical imaging of bone.

The global burden of bone-related diseases is increasing in the aging society; thus, improved bone targeted imaging for their early identification and treatment are needed. In this study, we screened novel peptide ligands for hydroxyapatite, a major inorganic component of teeth and bones, and identified a peptide enabling in vivo bone targeting and real-time fluorescence bone detection. To isolate peptides highly specific for hydroxyapatite, we used negative and positive selection from a randomized 8-mer peptide phage library and identified hydroxyapatite-specific peptides (HA-pep2, HA-pep3, and HA-pep7). Among these three peptides, HA-pep3 showed the highest binding capacity and superior dissociation constant towards hydroxyapatite surfaces over time (~ 88.3% retained on hydroxyapatite after two weeks). Furthermore, HA-pep3 was highly specific for hydroxyapatite compared to other calcium salt-based materials. Using this superior specificity, HA-pep3 showed higher accumulation in skull, spine, and joints in comparison with scrambled control peptide during real-time whole-body imaging. Ex vivo analysis of the major organs and bone from mice demonstrated that the fluorescence intensity in bone was about 3.32 folds higher in the case of HA-pep3 than the one exhibited by the scrambled control peptide. Our study identified a novel approach for targeting ligands for bone specific imaging and can be useful for drug delivery applications.

A Study of the Effects of Doxorubicin-Containing Liposomes on Osteogenesis of 3D Stem Cell Spheroids Derived from Gingiva.

The objective of the present investigation is to determine the effects of neutral, anionic, and cationic liposomes loaded with doxorubicin with thin-lipid-film-hydration method on the cellular viability and osteogenesis of stem cell spheroids. Spheroid formation and morphology of the three-dimensional spheroid were noted with an inverted microscope. Quantitative cellular viability was assessed using a commercially available kit. Osteogenic potential was evaluated by applying alkaline phosphatase activity and anthraquinone dye of Alizarin Red S. Western blot analysis was performed using collagen I expression. Spheroids were formed in each silicon elastomer-based concave microwell on Day 1. Noticeable changes of the spheroid were seen with a higher concentration of doxorubicin, especially in the cationic liposome group at Days 5 and 7. We found that the application of doxorubicin for 5 days significantly reduced the cellular viability. A higher concentration of doxorubicin produced a significant decrease in alkaline phosphatase activity. Alizarin Red S staining showed that extracellular calcium deposits were evenly noted in each group. An increase of calcium deposits was noted on Day 14 when compared to Day 7. The morphology of the groups with higher concentrations of doxorubicin showed to be more dispersed. We noticed that doxorubicin-loaded cationic liposomes resulted in the highest uptake of the examined cell spheroids and that doxorubicin-loaded liposomes affected the osteogenic differentiation. The implication of this study is that the type of liposome should be selected based on the purpose of the application.

Cellular viability and osteogenic differentiation potential of human gingiva-derived stem cells in 2D culture following treatment with anionic, cationic, and neutral liposomes containing doxorubicin.

The effects of doxorubicin, particularly doxorubicin liposome, on stem cells have remained to be fully elucidated. The aim of the present study was to evaluate the effects of anionic, cationic and neutral liposomes loaded with doxorubicin on the viability and osteogenic differentiation potential of human gingiva-derived stem cells in two-dimensional culture. Doxorubicin-loaded liposomes were prepared using the traditional thin-lipid-film hydration method. Stem cells were seeded on a culture plate and maintained in osteogenic media. The morphology of the stem cells was observed under an inverted microscope. The number of viable cells was determined using a Cell-Counting Kit-8 assay. The alkaline phosphatase activity was assessed and Alizarin Red S staining was performed to evaluate osteogenic differentiation. A higher concentration of doxorubicin caused noticeable changes in the morphology of the stem cells. Decreases in cellular viability were observed after applying doxorubicin. The application of doxorubicin, particularly at higher concentrations, produced a noticeable decrease in alkaline phosphatase activity and Alizarin Red S staining. The present study indicated that application of doxorubicin with or without liposomes reduced the cellular viability and osteogenic differentiation. Among the different treatments, the doxorubicin-loaded cationic liposomes induced the strongest reduction in the cellular viability and osteogenic differentiation in the stem cell culture.

Optimized phospholipid-based nanoparticles for inner ear drug delivery and therapy.

To develop efficient carriers for inner ear drug delivery, we prepared four kinds of phospholipid-based nanoparticles: neutral, anionic, cationic, and cationic-PEG (polyethyleneglycol) particles. PEG was used to maintain long-term particle circulation in the perilymph, avoiding non-specific binding of particles to proteins. All four nanoparticles were about 200 nm in diameter, and their zeta potentials were -4.32, -26.0, +25.8, and -0.28, respectively, for neutral, anionic, cationic, and cationic-PEG nanoparticles. To test particle efficacy in vitro, we used an artificial mucosa 100 µm in thickness to model the round window membrane (RWM) and HEI-OC1 cells, which were treated with particles containing Nile Red dye. Based on the levels of particle penetration and cellular uptake in this model system, we selected an optimal particle for further study. We also observed the movement of particles in ex vivo organotypic cultures of the organ of Corti. In mice, we analyzed the biodistribution of dexamethasone (Dex) in the inner ear after intratympanic injection of Dex-loaded nanoparticles. Then, we tested the therapeutic utility of the Dex-loaded nanoparticles in a mouse model of ototoxicity. In the auditory brainstem response (ABR) test, particle provided improved hearing loss recovery at all tested frequencies, more so than did the Dex sodium phosphate (Dex-SP) solution in current clinical use. Furthermore, quantitative PCR showed that nanoparticles reduced the levels of pro-inflammatory cytokines, exhibiting anti-inflammatory effects superior to those of Dex-SP. Thus, the surface properties of nanoparticles play pivotal roles in particle penetration and distribution after intratympanic injection. Our in vitro screening system using an artificial mucosa will also be valuable in the development of carriers for inner ear drug delivery.

Nano-sized metabolic precursors for heterogeneous tumor-targeting strategy using bioorthogonal click chemistry in vivo.

Herein, we developed nano-sized metabolic precursors (Nano-MPs) for new tumor-targeting strategy to overcome the intrinsic limitations of biological ligands such as the limited number of biological receptors and the heterogeneity in tumor tissues. We conjugated the azide group-containing metabolic precursors, triacetylated N-azidoacetyl-d-mannosamine to generation 4 poly(amidoamine) dendrimer backbone. The nano-sized dendrimer of Nano-MPs could generate azide groups on the surface of tumor cells homogeneously regardless of cell types via metabolic glycoengineering. Importantly, these exogenously generated 'artificial chemical receptors' containing azide groups could be used for bioorthogonal click chemistry, regardless of phenotypes of different tumor cells. Furthermore, in tumor-bearing mice models, Nano-MPs could be mainly localized at the target tumor tissues by the enhanced permeation and retention (EPR) effect, and they successfully generated azide groups on tumor cells in vivo after an intravenous injection. Finally, we showed that these azide groups on tumor tissues could be used as 'artificial chemical receptors' that were conjugated to bioorthogonal chemical group-containing liposomes via in vivo click chemistry in heterogeneous tumor-bearing mice. Therefore, overall results demonstrated that our nano-sized metabolic precursors could be extensively applied to new alternative tumor-targeting technique for molecular imaging and drug delivery system, regardless of the phenotype of heterogeneous tumor cells.

DNA amplification in neutral liposomes for safe and efficient gene delivery.

In general, traditional gene carriers contain strong cationic charges to efficiently load anionic genes, but this cationic character also leads to destabilization of plasma membranes and causes severe cytotoxicity. Here, we developed a PCR-based nanofactory as a safe gene delivery system. A few template plasmid DNA can be amplified by PCR inside liposomes about 200 nm in diameter, and the quantity of loaded genes highly increased by more than 8.8-fold. The liposome membrane was composed of neutral lipids free from cationic charges. Consequently, this system is nontoxic, unlike other traditional cationic gene carriers. Intense red fluorescent protein (RFP) expression in CHO-K1 cells showed that the amplified genes could be successfully transfected to cells. Animal experiments with the luciferase gene also showed in vivo gene expression by our system without toxicity. We think that this PCR-based nanofactory system can overcome the toxicity problem that is the critical limitation of current gene delivery to clinical application.

In vivo NIRF imaging of tumor targetability of nanosized liposomes in tumor-bearing mice.

To optimize tumor targetability of nanosized liposomes for application as drug carriers, various liposomes are prepared by incorporating different amounts (10, 30, and 50 wt%) of cationic, anionic, and PEGylated lipids into neutral lipid. In vivo near-infrared fluorescence images reveal that PEG-PE/PC liposomes display high tumor accumulation in tumor-bearing mice, while large amounts of DOTAP/PC liposomes are rapidly captured in the liver, resulting in poor tumor accumulation. These results demonstrate that optimization of the surface properties of liposomes is very important for their tumor targetability, and that in vivo imaging techniques are useful in developing and optimizing nanosized liposome-based drug carriers.

Polyethylene glycol-conjugated hyaluronic acid-ceramide self-assembled nanoparticles for targeted delivery of doxorubicin.

Polyethylene glycol (PEG)-conjugated hyaluronic acid-ceramide (HACE) was synthesized for the preparation of doxorubicin (DOX)-loaded HACE-PEG-based nanoparticles, 160 nm in mean diameter with a negative surface charge. Greater uptake of DOX from these HACE-PEG-based nanoparticles was observed in the CD44 receptor highly expressed SCC7 cell line, compared to results from the CD44-negative cell line, NIH3T3. A strong fluorescent signal was detected in the tumor region upon intravenous injection of cyanine 5.5-labeled nanoparticles into the SCC7 tumor xenograft mice; the extended circulation time of the HACE-PEG-based nanoparticle was also observed. Pharmacokinetic study in rats showed a 73.0% reduction of the in vivo clearance of DOX compared to the control group. The antitumor efficacy of the DOX-loaded HACE-PEG-based nanoparticles was also verified in a tumor xenograft mouse model. DOX was efficiently delivered to the tumor site by active targeting via HA and CD44 receptor interaction and by passive targeting due to its small mean diameter (<200 nm). Moreover, PEGylation resulted in prolonged nanoparticle circulation and reduced DOX clearance rate in an in vivo model. These results therefore indicate that PEGylated HACE nanoparticles represent a promising anticancer drug delivery system for cancer diagnosis and therapy.

Gas-generating polymeric microspheres for long-term and continuous in vivo ultrasound imaging.

Ultrasound (US) imaging is one of the most common biomedical imaging methods, due to the easy assessment and noninvasive way. For more precise and accurate US imaging, many contrast agents have been developed in a form of microbubbles composed of inner gas and shell materials. However, microbubbles showed undesirable short half-life under acoustic field during US imaging and insufficient in vivo stability in blood flow due to diffusion or bubble destruction. Therefore, the improvement of the half-life and stability of microbubbles under in vivo condition is highly needed for long-term in vivo US imaging. Herein, we developed rationally designed gas-generating polymeric microsphere (GGPM) that can produce microbubbles without encapsulation of gas for long-term and continuous US imaging. The poly(cholesteryl γ-butyrolactone-b-propylene oxide), poly(CB-PO), with carbonate side chains was synthesized as gas-generating polymer by ring-opening polymerization of cholestryl γ-butyrolactone (CB) and propylene oxide (PO). As optimal structure for intense US signal generation, porous GGPMs (p-GGPMs) with the average size about 3-5 µm were prepared with poly(CB-PO) by double emulsion method. These p-GGPMs generated continuous US signals over 70 min, while the signals from Sonovue(®), a commercial US contrast agent were completely attenuated within 15 min. This long-term signal duration of p-GGPM was also reproduced when they were subcutaneously injected under the skin of mouse. Moreover, as advanced in vivo application, the fine US imaging of heart in rat was enabled by intravenous injection of p-GGPM. Therefore, these overall results showed the great potential of p-GGPM as gas-generating US contrast agent for in vivo biomedical imaging and diagnosis.