Mucoadhesive Hybrid System of Silk Fibroin Nanoparticles and Thermosensitive In Situ Hydrogel for Amphotericin B Delivery: A Potential Option for Fungal Keratitis Treatment.

The purpose of this work was to investigate the feasibility of a novel ophthalmic formulation of amphotericin B-encapsulated silk fibroin nanoparticles incorporated in situ hydrogel (AmB-FNPs ISG) for fungal keratitis (FK) treatment. AmB-FNPs ISG composites were successfully developed and have shown optimized physicochemical properties for ocular drug delivery. Antifungal effects against Candida albicans and in vitro ocular irritation using corneal epithelial cells were performed to evaluate the efficacy and safety of the composite formulations. The combined system of AmB-FNPs-ISG exhibited effective antifungal activity and showed significantly less toxicity to HCE cells than commercial AmB. In vitro and ex vivo mucoadhesive tests demonstrated that the combination of silk fibroin nanoparticles with in situ hydrogels could enhance the adhesion ability of the particles on the ocular surface for more than 6 h, which would increase the ocular retention time of AmB and reduce the frequency of administration during the treatment. In addition, AmB-FNP-PEG ISG showed good physical and chemical stability under storage condition for 90 days. These findings indicate that AmB-FNP-PEG ISG has a great potential and be used in mucoadhesive AmB eye drops for FK treatment.

Crosslinked Fibroin Nanoparticles: Investigations on Biostability, Cytotoxicity, and Cellular Internalization.

Recently, crosslinked fibroin nanoparticles (FNP) using the crosslinker 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) or the polymer poly(ethylenimine) (PEI) have been developed and showed potentials as novel drug delivery systems. Thus, this study further investigated the biological properties of these crosslinked FNP by labeling them with fluorescein isothiocyanate (FITC) for in vitro studies. All formulations possessed a mean particle size of approximately 300 nm and a tunable zeta potential (-20 to + 30 mV) dependent on the amount/type of crosslinkers. The FITC-bound FNP showed no significant difference in physical properties compared to the blank FNP. They possessed a binding efficacy of 3.3% w/w, and no FITC was released in sink condition up to 8 h. All formulations were colloidal stable in the sheep whole blood. The degradation rate of these FNP in blood could be controlled depending on their crosslink degree. Moreover, no potential toxicity in erythrocytes, Caco-2, HepG2, and 9L cells was noted for all formulations at particle concentrations of < 1 mg/mL. Finally, all FNP were internalized into the Caco-2 cells after 3 h incubation. The uptake rate of the positively charged particles was significantly higher than the negatively charged ones. In summary, the crosslinked FNP were safe and showed high potentials as versatile systems for biomedical applications.

Paclitaxel loaded EDC-crosslinked fibroin nanoparticles: a potential approach for colon cancer treatment.

Colon cancer is one of the most life-threatening cancers with high incidence and mortality rates. Current first-line treatments are ineffective and possess many unwanted effects. The off-label use of paclitaxel encapsulated in nanoparticles proves an innovative approach. In this study, we reported novel paclitaxel loaded EDC-crosslinked fibroin nanoparticles (PTX-FNPs) for anticancer purpose. The particles were formulated using desolvation method and the physicochemical properties were controlled favorably, including the particle size (300-500 nm), zeta potential (- 15 to + 30 mV), drug entrapment efficiency (75-100%), crystallinity, drug solubility (1- to 10-fold increase), dissolution profiles, stability (> 24 h in intravenous diluent and > 6 months storage at 4 °C). In in vitro study, all formulations showed no toxicity on the red blood cells, whereas retained the paclitaxel cytotoxicity on MCF-7 breast cancer and Caco-2 colon cancer cells. Interestingly, PTX-FNPs can be uptaken rapidly by the Caco-2 cells, consequently increased paclitaxel potency up to 10-fold compared to the free drug. Graphical abstract.

Alpha mangostin loaded crosslinked silk fibroin-based nanoparticles for cancer chemotherapy.

Silk fibroin has been utilized extensively for biomedical purposes, especially the drug delivery systems. This study introduced and characterized three novel α-mangostin loaded crosslinked fibroin nanoparticles (FNPs), using EDC or PEI as a crosslinker, for cancer treatment. All three formulas were spherical particles with a mean size of approximately 300 nm. By varying the type and/or amount of the crosslinkers, particle surface charge was controllable from -15 to +30 mV. Crosslinked FNPs exhibited higher drug entrapment efficiency (70%) and drug loading (7%) than non-crosslinked FNP. FT-IR, XRD, and DSC analytical methods confirmed that α-mangostin was entrapped in FNPs in molecular dispersion form. Compared to the free α-mangostin, the crosslinked FNPs increased the drug's solubility up to threefold. They also showed sustained release characteristics of more than 3 days, and reduced free α-mangostin hematotoxicity by 90%. The α-mangostin loaded FNPs were physicochemically stable for up to 24 h when dispersed in intravenous diluent and for at least 6 months when preserved as lyophilized powder at 4 °C. In terms of anticancer efficacy, on both Caco-2 colorectal and MCF-7 breast adenocarcinoma cell lines, all formulas maintain α-mangostin's apoptotic effect while exhibit greater cytotoxicity than the free drug. In conclusion, α-mangostin loaded crosslinked FNPs show high potential for cancer chemotherapy.

The trifunctional protein mediates thyroid hormone receptor-dependent stimulation of mitochondria metabolism.

We previously demonstrated that the thyroid hormone, T(3), acutely stimulates mitochondrial metabolism in a thyroid hormone receptor (TR)-dependent manner. T(3) has also recently been shown to stimulate mitochondrial fatty acid oxidation (FAO). Here we report that TR-dependent stimulation of metabolism is mediated by the mitochondrial trifunctional protein (MTP), the enzyme responsible for long-chain FAO. Stimulation of FAO was significant in cells that expressed a nonnuclear amino terminus shortened TR isoform (sTR(43)) but not in adult fibroblasts cultured from mice deficient in both TRα and TRß isoforms (TRα(-/-)ß(-/-)). Mouse embryonic fibroblasts deficient in MTP (MTP(-/-)) did not support T(3)-stimulated FAO. Inhibition of fatty-acid trafficking into mitochondria using the AMP-activated protein kinase inhibitor 6-[4-(2-piperidin-1-yl-ethoxy)-phenyl)]-3-pyridin-4-yl-pyrrazolo[1,5-a]-pyrimidine (compound C) or the carnitine palmitoyltransferase 1 inhibitor etomoxir prevented T(3)-stimulated FAO. However, T(3) treatment could increase FAO when AMP-activated protein kinase was maximally activated, indicating an alternate mechanism of T(3)-stimulated FAO exists, even when trafficking is presumably high. MTPα protein levels and higher molecular weight complexes of MTP subunits were increased by T(3) treatment. We suggest that T(3)-induced increases in mitochondrial metabolism are at least in part mediated by a T(3)-shortened TR isoform-dependent stabilization of the MTP complex, which appears to lower MTP subunit turnover.

Inhibition of apoptotic potency by ligand stimulated thyroid hormone receptors located in mitochondria.

We recently reported that shortened thyroid hormone receptor isoforms (TRs) can target mitochondria and acutely modulate inositol 1,4,5 trisphosphate (IP3)-mediated Ca2+ signaling when activated by thyroid hormone 3,5,3'-tri-iodothyronine (T3). Stimulation occurs via an increase in mitochondrial metabolism that is independent of transcriptional activity. Here, we present evidence that T3-bound xTRbetaA1s inhibit apoptotic activity mediated by cytochrome c release. An assay for apoptotic potency was modified to measure the ability of Xenopus oocyte extracts to induce morphological changes in isolated liver nuclei. Apoptotic potency was significantly decreased when oocyte extract was prepared from xTRbetaA1 expressing oocytes and treated with T3. The ability of T3 treatment to inhibit apoptosis was dependent on the expression of xTRbetaA1s in the mitochondrial fraction, not in the cytosolic fraction. T3 treatment also increased the membrane potential of isolated mitochondria prepared from oocytes expressing xTRbetaA1s but not from wildtype controls. We conclude that T3 acutely regulates cytochrome c release in a potential dependent manner by activating TRs located within mitochondria.

Nontranscriptional modulation of intracellular Ca2+ signaling by ligand stimulated thyroid hormone receptor.

Thyroid hormone 3,5,3'-tri-iodothyronine (T3) binds and activates thyroid hormone receptors (TRs). Here, we present evidence for a nontranscriptional regulation of Ca2+ signaling by T3-bound TRs. Treatment of Xenopus thyroid hormone receptor beta subtype A1 (xTRbetaA1) expressing oocytes with T3 for 10 min increased inositol 1,4,5-trisphosphate (IP3)-mediated Ca2+ wave periodicity. Coexpression of TRbetaA1 with retinoid X receptor did not enhance regulation. Deletion of the DNA binding domain and the nuclear localization signal of the TRbetaA1 eliminated transcriptional activity but did not affect the ability to regulate Ca2+ signaling. T3-bound TRbetaA1 regulation of Ca2+ signaling could be inhibited by ruthenium red treatment, suggesting that mitochondrial Ca2+ uptake was required for the mechanism of action. Both xTRbetaA1 and the homologous shortened form of rat TRalpha1 (rTRalphaDeltaF1) localized to the mitochondria and increased O2 consumption, whereas the full-length rat TRalpha1 did neither. Furthermore, only T3-bound xTRbetaA1 and rTRalphaDeltaF1 affected Ca2+ wave activity. We conclude that T3-bound mitochondrial targeted TRs acutely modulate IP3-mediated Ca2+ signaling by increasing mitochondrial metabolism independently of transcriptional activity.