CircTBX5 knockdown modulates the miR-558/MyD88 axis to alleviate IL-1ß-induced inflammation, apoptosis and extracellular matrix degradation in chondrocytes via inactivating the NF-κB signaling.

BACKGROUND: It has been widely shared that the dysregulation of circular RNA (circRNA) may contribute to the progression of osteoarthritis (OA). OA is characterized by persistent chondrocyte injury. We aimed to clarify the role of circTBX5 in IL-1ß-induced chondrocyte injury. METHODS: The expression of circTBX5, miR-558 and MyD88 mRNA was measured using quantitative real-time PCR (qPCR). Cell viability, proliferation and apoptosis were assessed by CCK-8, EdU or flow cytometry assay. The protein levels of extracellular matrix (ECM)-associated markers, MyD88, IkBα, p65 and phosphorylated IkBα were measured by western blot. The release of inflammatory factors was assessed by ELISA. The targets of circTBX5 were screened by RIP and pull-down assay. The putative binding between miR-558 and circTBX5 or MyD88 was validated by dual-luciferase reporter assay. RESULTS: CircTBX5 and MyD88 were enhanced, while miR-558 was downregulated in OA cartilage tissues and IL-1ß-treated C28/I2 cells. IL-1ß induced C28/I2 cell injury by impairing cell viability and proliferation and promoting cell apoptosis, ECM degradation and inflammatory response, while circTBX5 knockdown alleviated IL-1ß induced injury. CircTBX5 bound to miR-558 to regulate IL-1ß induced cell injury. In addition, MyD88 was a target of miR-558, and circTBX5 targeted miR-558 to positively regulate MyD88 expression. MiR-558 enrichment attenuated IL-1ß induced injury by sequestering MyD88 expression. Moreover, circTBX5 knockdown weakened the activity of NF-κB signaling, while miR-558 inhibition or MyD88 overexpression recovered the activity of NF-κB signaling. CONCLUSION: CircTBX5 knockdown modulated the miR-558/MyD88 axis to alleviate IL-1ß induced chondrocyte apoptosis, ECM degradation and inflammation via inactivating the NF-кB signaling pathway.

Synergy effects of copper ion in doxorubicin-based chelate prodrug for cancer chemo-chemodynamic combination therapy.

Doxorubicin (DOX) is a commonly studied chemotherapeutic agent for the treatment of solid tumors, but the severe side effects limit its clinical application. It is shown that DOX-metal chelate has lower in vitro cytotoxicity compared with DOX, as the anthracyclines of DOX can form coordinative interaction with transition metal ions. In addition, the transition metal ions could catalyze the production of hydroxyl radicals (·OH) via Fenton/Fenton-like reactions to achieve antitumor chemodynamic therapy (CDT). In this study, copper ions (Cu2+) were applied to obtain DOX/Cu(II) prodrug, and a liposomal formulation was used to avoid the rapid blood clearance and optimize the biodistribution of this prodrug. In vitro and in vivo antitumor results demonstrated that this pH sensitive Cu-chelating prodrug can reduce adverse effects of DOX but improve the antitumor efficiency due to the combination of chemotherapy and chemodynamic therapy. Our study provided a facile and effective approach of metal-chelating prodrug strategy for combination cancer therapy strategy.

Hypoxia responsive nano-drug delivery system based on angelica polysaccharide for liver cancer therapy.

Based on the tumor hypoxic microenvironment and the new programmed cell death mode of combined ferroptosis, an angelica polysaccharide-based nanocarrier material was synthesized. The polymer contains hydrophilic angelica polysaccharide (ASP) that is linked by azobenzene (AZO) linker with ferrocene (Fc), and then the side chain was covalently modified with arachidonic acid (AA). It was postulated that the polymer micelles could work as an instinctive liver targeting drug delivery carrier, owing to the existence of ASP with liver targeting. Moreover, the aim was to engineer hypoxia-responsive polymer micelles which was modified by AA, for selective enhancement of ferroptosis in solid tumor, via diminishing glutathione (GSH) under hypoxia. Finally, we synthesized the amphiphilic polymer micelles AA/ASP-AZO-Fc (AAAF) by self-assembling. The structure of AAAF was confirmed by 1H-NMR and FT-IR. Then, we exemplified the hydrophobic medication curcumin into polymer micelles AAAF@Cur, which has smooth and regular spheres. In vitro release test affirmed that AAAF@Cur can achieve hypoxia response to drug release. In addition, a series of cell experiments confirmed that hypoxia could enhance cell uptake and effectively improve the proliferation inhibitory activity of HepG2 cells. In conclusion, AAAF, as an effective cell carrier, is expected to develop in sensitizing ferroptosis and anti-tumor.

Novel Mitochondrial Targeting Multifunctional Surface Charge-Reversal Polymeric Nanoparticles for Cancer Treatment.

Polymeric nanoparticles were widely used as delivery vehicles for targeted delivery of anticancer drugs, because of their targeting property and versatility. Mitochondria are one of the important organelles that regulate the apoptosis of cancer cells and can be considered as a pivotal target for cancer treatment. A pH-responsive charge-reversal and mitochondrial targeting nanoparticles, Vitamin B6-oligomeric hyaluronic acid-dithiodipropionic acid-berberine (B6-oHA-SS-Ber), were prepared in this study. Ber is a lipophilic cation that was conjugated with oHA through disulfide bonds to produce mitochondria-targeted conjugates (oHA-SS-Ber). B6 was conjugated to oHA to obtain B6-oHA-SS-Ber and the two types of Cur-loaded nanoparticles (Cur-NPs) were formulated by the dialysis method. Due to pKa of B6, the charge they carried in the tumor tissue acidic microenvironment can be transferred from negative charge to positive charge, further targeting mitochondria. In our study, we successfully synthesized B6-HA-SS-Ber and characterized the structure by 1H-NMR. According to the results of transmission electron microscopy (TEM), we found that the B6-oHA-SS-Ber/Cur micelles could self-assembled in water to form spherical nanoparticles, with a hydrodynamic diameter of 172.9±13 nm. Moreover, in vitro cytotoxicity, cellular uptake, lysosome escape and mitochondrial distribution researches revealed the better effect of B6-oHA-SS-Ber/Cur micelles in comparison to oHA-SS-Ber/Cur. In vivo anticancer activities indicated that the B6-oHA-SS-Ber/Cur micelles exhibited effective inhibition of tumor growth.

Afatinib-loaded immunoliposomes functionalized with cetuximab: A novel strategy targeting the epidermal growth factor receptor for treatment of non-small-cell lung cancer.

Afatinib, a selective and irreversible inhibitor of tyrosine kinase, was approved for the treatment of advanced non-small-cell lung cancer (NSCLC) with epidermal growth factor receptor (EGFR) overexpression in 2013. Cetuximab (CTX), an anti-EGFR monoclonal antibody, is co-administered with afatinib to improve efficacy. Unfortunately, dose-related adverse reactions caused by combination therapy have affected patient compliance, and have resulted in treatment discontinuation in severe cases. In the present study, afatinib was encapsulated in "liposomes" (LPs) to achieve longer circulation in the blood and an enhanced permeability-and-retention effect in tumors. Concomitantly, CTX was designed to bind to drug-loaded LPs to form "immuno-LPs" for tumor-cell selectivity and therapeutic activity. In vitro, the cellular internalization rate of immuno-LPs was significantly higher than that of LPs (p < 0.05). In vivo, a markedly increased area under the curve and prolonged terminal half-life were detected in rats injected with the two LP formulations, indicating that LP encapsulation protected afatinib from binding to hemoglobin to control the risk of idiosyncratic drug reactions. Compared with free afatinib and LPs, immuno-LPs exhibited strongly enhanced drug delivery and antitumor efficacy in an NSCLC xenograft model, with stronger tumor selectivity and potentially fewer side-effects. Hence, EGFR-targeting immuno-LPs appear to be promising for NSCLC treatment.

Entecavir-loaded poly (lactic-co-glycolic acid) microspheres for long-term therapy of chronic hepatitis-B: Preparation and in vitro and in vivo evaluation.

To avoid severe exacerbations in the load of hepatitis B virus (HBV) as a consequence of discontinuous use of anti-HBV drugs, entecavir (ETV), the first-line anti-HBV drug, was primally formulated as extended-release poly (lactic-co-glycolic acid) microspheres in the present study. Because ETV is slightly soluble in water and in some other organic solvents used for microsphere preparation, methods for solid-microencapsulation were employed to fabricate the ETV microspheres. The optimized microspheres were evaluated for their morphology, particle size, drug loading, in vitro drug release, and in vivo pharmacokinetics in rats. The optimized formulation was found to have a mean particle size of 86 µm and drug loading of 13%. Differential scanning calorimetry and powder X-ray diffraction indicated that ETV existed in crystal, amorphous, and molecular states in the microspheres. In vitro and in vivo release revealed that the dissolution of ETV dominated the release process. The morphology of the microspheres and changes in the morphology during in vitro release were assessed by scanning electron microscopy. The novel ETV-MS described in this study should have great potential for clinical use as an alternative treatment against HBV.

Synthesis of CSK-DEX-PLGA Nanoparticles for the Oral Delivery of Exenatide to Improve Its Mucus Penetration and Intestinal Absorption.

The oral absorption of exenatide, a drug for type 2 diabetes treatment, can be improved by using nanoparticles (NPs) for its delivery. To improve the mucus penetration and intestinal absorption of exenatide, we designed a block copolymer, CSKSSDYQC-dextran-poly(lactic-co-glycolic acid) (CSK-DEX-PLGA), and used it for the preparation of exenatide-loaded NPs. The functionalized exenatide-loaded NPs composed of CSK-DEX-PLGA were able to target intestinal epithelial cells and reduce the mucus-blocking effect of the intestine. Moreover, the CSK modification of DEX-PLGA was found to significantly promote the absorption efficiency of NPs in the small intestine based on in vitro ligation of the intestinal rings and an examination of different intestinal absorption sites. Compared to DEX-PLGA-NPs (DPs), the absorption of CSK-DEX-PLGA-NPs (CDPs) was increased in the villi, allowing the drug to act on gobletlike Caco-2 cells through clathrin-, caveolin-, and gap-mediated endocytosis. Furthermore, the enhanced transport ability of CDPs was observed in a study on Caco-2/HT-29-MTX cocultured cells. CDPs exhibited a prolonged hypoglycemic response with a relative bioavailability of 9.2% in diabetic rats after oral administration. In conclusion, CDPs can target small intestinal goblet cells and have a beneficial effect on the oral administration of macromolecular peptides as a nanometer-sized carrier.

Nose-to-brain delivery of temozolomide-loaded PLGA nanoparticles functionalized with anti-EPHA3 for glioblastoma targeting.

Glioblastoma is the most common malignant brain tumor. Efficient delivery of drugs targeting glioblastomas remains a challenge. Ephrin type-A receptor 3 (EPHA3) tyrosine kinase antibody-modified polylactide-co-glycolide (PLGA) nanoparticles (NPs) were developed to target glioblastoma via nose-to-brain delivery. Anti-EPHA3-modified, TBE-loaded NPs were prepared using an emulsion-solvent evaporation method, showed a sustained in vitro release profile up to 48 h and a mean particle size of 145.9 ± 8.7 nm. The cellular uptake of anti-EPHA3-modified NPs by C6 cells was significantly enhanced compared to that of nontargeting NPs (p < .01). In vivo imaging and distribution studies on the glioma-bearing rats showed that anti-EPHA3-modified NPs exhibited high fluorescence intensity in the brain and effectively accumulated to glioma tissues, indicating the targeting effect of anti-EPHA3. Glioma-bearing rats treated with anti-EPHA3-modified NPs resulted in significantly higher tumor cell apoptosis (p < .01) than that observed with other formulations and prolonged the median survival time of glioma-bearing rats to 26 days, which was 1.37-fold longer than that of PLGA NPs. The above results indicated that anti-EPHA3-modified NPs may potentially serve as a nose-to-brain drug carrier for the treatment of glioblastoma.

Multivesicular liposomes for sustained release of bevacizumab in treating laser-induced choroidal neovascularization.

Bevacizumab is an anti-vascular endothelial growth factor drug that can be used to treat choroidal neovascularization (CNV). Bevacizumab-loaded multivesicular liposomes (Bev-MVLs) have been designed and developed to increase the intravitreal retention time of bevacizumab and reduce the number of injection times. In this study, Bev-MVLs with high encapsulation efficiency were prepared by double emulsification technique, and antibody activity was determined. The results revealed that 10% of human serum albumin (HSA) could preserve the activity of bevacizumab. In vitro release of Bev-MVLs appeared to be in a more sustained manner, the underlying mechanisms of Bev-MVLs indicated that bevacizumab was released from MVLs through diffusion and erosion. Results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) demonstrated that bevacizumab could retain its structural integrity after being released from MVLs in vitro. In vivo imaging was used to evaluate the retention time of antibody in rat eyes, while pharmacokinetic analysis was performed on rabbit eyes. These results indicated that Bev-MVLs exhibited sustained release effects as compared to bevacizumab solution (Bev-S). Bev-MVLs could effectively inhibit the thickness of CNV lesion as compared to Bev-S at 28 days after treatment. Furthermore, these data suggest that Bev-MVLs are biologically feasible to increase the retention time of bevacizumab in vitreous humor. This novel Bev-MVLs may therefore serve as a promising sustained release drug delivery system for the treatment of CNV.

Development, validation and comparison of surrogate matrix and surrogate analyte approaches with UHPLC-MS/MS to simultaneously quantify dopamine, serotonin and γ-aminobutyric acid in four rat brain regions.

As biomarkers, endogenous neurotransmitters play critical roles in the process of neuropsychiatric diseases, and neurotransmitter levels in different brain regions can contribute to neurological disease diagnosis and treatment. Due to the lack of a blank matrix for endogenous neurotransmitters, surrogate-matrix and surrogate-analyte approaches have been used for the determination of neurotransmitters to solve this problem. In this study, we capitalised on the high accuracy, precision, and throughput of UHPLC-MS/MS and developed new methods based on the two approaches. Both approaches satisfied FDA and EMA validation criterias after an appropriate parallelism assessment, and they were used to further quantify the three endogenous neurotransmitters, including dopamine (DA), serotonin (5-HT) and γ-aminobutyric acid (GABA) in rat brain four regions (cortex, striatum, hypothalamus and hippocampus) which represent the catecholamines, indolamines, and amino acids, respectively. Comparison of the results in the same rats (n = 10) showed there was no significant difference in DA, 5-HT, or GABA levels between the two approaches (P > 0.05). The concentrations of DA and GABA were highest in striatum and hypothalamus, respectively, and the levels of 5-HT were paralleled in striatum and hippocampus almost 2-fold higher than other regions. This is the first study to compare these two approaches in the determination of endogenous neurotransmitter content in the rat brain, and the surrogate-matrix approach proved to be simple, rapid, and reliable, considering cost, matrix similarity, and practicality.

Trastuzumab- and Fab' fragment-modified curcumin PEG-PLGA nanoparticles: preparation and evaluation in vitro and in vivo.

INTRODUCTION: Nanoparticles (NPs) modified with bio-ligands represent a promising strategy for active targeted drug delivery to tumour. However, many targeted ligands, such as trastuzumab (TMAB), have high molecular weight, limiting their application for targeting. In this study, we prepared Fab' (antigen-binding fragments cut from TMAB)-modified NPs (Fab'-NPs) with curcumin (Cur) as a model drug for more effective targeting of human epidermal growth factor receptor 2 (HER2/ErbB2/Neu), which is overexpressed on breast cancer cells. MATERIAL AND METHODS: The release kinetics was conducted by dialysis bags. The ability to kill HER2-overexpressing BT-474 cells of Fab'-Cur-NPs compared with TMAB-Cur-NPs was conducted by cytotoxicity experiments. Qualitative and quantitative cell uptake studies using coumarin-6 (fluorescent probe)-loaded NPs were performed by fluorescence microscopy and flow cytometry. Pharmacokinetics and biodistribution experiments in vivo were assessed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). RESULTS: The release kinetics showed that both Fab'-Cur-NPs and TMAB-Cur-NPs provided continuous, slow release of curcumin for 72 h, with no significant difference. In vitro cytotoxicity experiments showed that Fab'-Cur-NPs manifested prominent ability to kill HER2-overexpressing BT-474 cells compared with TMAB-Cur-NPs. Qualitative and quantitative cell uptake studies indicated that the accumulation of Fab'-NPs was greater than that of TMAB-NPs in BT-474 (HER2+) cells; However, there was no significant difference in MDA-MB-231 (HER2-) cells. Pharmacokinetics and biodistribution experiments in vivo demonstrated that the half-life (t1/2) and area under the blood concentration-time curve (AUC0-t) of Fab'-Cur-NPs increased 5.30-fold and 1.76-fold relative to those of TMAB-Cur-NPs, respectively. Furthermore, the tumor accumulation of Fab'-Cur-NPs was higher than that of TMAB-Cur-NPs. CONCLUSION: Fab' fragment has greater capacity than the intact antibody to achieve tumor targeting through NP-based delivery.

RVG29-modified docetaxel-loaded nanoparticles for brain-targeted glioma therapy.

Gliomas are the most common malignant brain tumor, but treatment is limited by the blood-brain barrier (BBB), especially for chemotherapeutic drugs. Although some chemotherapy drugs can pass through the BBB, many of these agents are toxic to normal brain tissue. To maximize therapeutic effects, chemotherapeutic drugs must accumulate at the glioma site. In this study, a specific ligand (the RVG29 peptide) that can combine with acetylcholine receptors was conjugated to polyethylene glycol-modified poly-(d,l-lactide-co-glycolide) (PEG-PLGA) to develop a targeted carrier; preparation of the targeted docetaxel nanoparticles (DTX-NPs) was performed by the nanoprecipitation method. The NPs were approximately 110 nm and had smooth surfaces. Enzyme-linked immunoassay results showed that the amount of receptor on the surface of glioma cells was 2.04-fold higher than that of nonmalignant cells, which may promote accumulation of RVG29-modified NPs at the targeting site. NPs showed targeting properties for glioma cells compared with the non-targeting NPs in an in vitro cellular uptake test. Targeted NPs also showed better BBB penetration in an in vitro model. In vivo tests indicated that RVG29-PEG-PLGA-NPs could selectively accumulate in intracranial glioma tissue. In conclusion, these results indicated that the RVG29-modified NPs have potential efficacy for glioma therapy.

Intranasal delivery of Huperzine A to the brain using lactoferrin-conjugated N-trimethylated chitosan surface-modified PLGA nanoparticles for treatment of Alzheimer's disease.

BACKGROUND: Safe and effective delivery of therapeutic drugs to the brain is important for successful therapy of Alzheimer's disease (AD). PURPOSE: To develop Huperzine A (HupA)-loaded, mucoadhesive and targeted polylactide-co-glycoside (PLGA) nanoparticles (NPs) with surface modification by lactoferrin (Lf)-conjugated N-trimethylated chitosan (TMC) (HupA Lf-TMC NPs) for efficient intranasal delivery of HupA to the brain for AD treatment. METHODS: HupA Lf-TMC NPs were prepared using the emulsion-solvent evaporation method and optimized using the Box-Behnken design. The particle size, zeta potential, drug entrapment efficiency, adhesion and in vitro release behavior were investigated. The cellular uptake was investigated by fluorescence microscopy and flow cytometry. MTT assay was used to evaluate the cytotoxicity of the NPs. In vivo imaging system was used to investigate brain targeting effect of NPs after intranasal administration. The biodistribution of Hup-A NPs after intranasal administration was determined by liquid chromatography-tandem mass spectrometry. RESULTS: Optimized HupA Lf-TMC NPs had a particle size of 153.2±13.7 nm, polydispersity index of 0.229±0.078, zeta potential of +35.6±5.2 mV, drug entrapment efficiency of 73.8%±5.7%, and sustained release in vitro over a 48 h period. Adsorption of mucin onto Lf-TMC NPs was 86.9%±1.8%, which was significantly higher than that onto PLGA NPs (32.1%±2.5%). HupA Lf-TMC NPs showed lower toxicity in the 16HBE cell line compared with HupA solution. Qualitative and quantitative cellular uptake experiments indicated that accumulation of Lf-TMC NPs was higher than nontargeted analogs in 16HBE and SH-SY5Y cells. In vivo imaging results showed that Lf-TMC NPs exhibited a higher fluorescence intensity in the brain and a longer residence time than nontargeted NPs. After intranasal administration, Lf-TMC NPs facilitated the distribution of HupA in the brain, and the values of the drug targeting index in the mouse olfactory bulb, cerebrum (with hippocampus removal), cerebellum, and hippocampus were about 2.0, 1.6, 1.9, and 1.9, respectively. CONCLUSION: Lf-TMC NPs have good sustained-release effect, adhesion and targeting ability, and have a broad application prospect as a nasal drug delivery carrier.

Preparation and evaluation of injectable Rasagiline mesylate dual-controlled drug delivery system for the treatment of Parkinson's disease.

A microsphere-gel in situ forming implant (MS-Gel ISFI) dual-controlled drug delivery system was applied to a high water-soluble small-molecule compound Rasagiline mesylate (RM) for effective treatment of Parkinson's disease. This injectable complex depot system combined an in situ phase transition gel with high drug-loading and encapsulation efficiency RM-MS prepared by a modified emulsion-phase separation method and optimized by Box-Behnken design. It was evaluated for in vitro drug release, in vivo pharmacokinetics, and in vivo pharmacodynamics. We found that the RM-MS-Gel ISFI system showed no initial burst release and had a long period of in vitro drug release (60 days). An in vivo pharmacokinetic study indicated a significant reduction (p < .01) in the initial high plasma drug concentration of the RM-MS-Gel ISFI system compared to that of the single RM-MS and RM-in situ gel systems after intramuscular injection to rats. A pharmacodynamic study demonstrated a significant reduction (p < .05) in 6-hydroxydopamine-induced contralateral rotation behavior and an effective improvement (p < .05) in dopamine levels in the striatum of the lesioned side after 28 days in animals treated with the RM-MS-Gel ISFI compared with that of animals treated with saline. MS-embedded in situ phase transition gel is superior for use as a biodegradable and injectable sustained drug delivery system with a low initial burst and long period of drug release for highly hydrophilic small molecule drugs.

Topical ocular delivery to laser-induced choroidal neovascularization by dual internalizing RGD and TAT peptide-modified nanoparticles.

A nanoparticle (NP) was developed to target choroidal neovascularization (CNV) via topical ocular administration. The NPs were prepared through conjugation of internalizing arginine-glycine-aspartic acid RGD (iRGD; Ac-CCRGDKGPDC) and transactivated transcription (TAT) (RKKRRQRRRC) peptide to polymerized ethylene glycol and lactic-co-glycolic acid. The iRGD sequence can specifically bind with integrin αvß3, while TAT facilitates penetration through the ocular barrier. 1H nuclear magnetic resonance and high-performance liquid chromatography demonstrated that up to 80% of iRGD and TAT were conjugated to poly(ethylene glycol)- poly(lactic-co-glycolic acid). The resulting particle size was 67.0±1.7 nm, and the zeta potential of the particles was -6.63±0.43 mV. The corneal permeation of iRGD and TAT NPs increased by 5.50- and 4.56-fold compared to that of bare and iRGD-modified NPs, respectively. Cellular uptake showed that the red fluorescence intensity of iRGD and TAT NPs was highest among primary NPs and iRGD- or TAT-modified NPs. CNV was fully formed 14 days after photocoagulation in Brown Norway (BN) rats as shown by optical coherence tomography and fundus fluorescein angiography analyses. Choroidal flat mounts in BN rats showed that the red fluorescence intensity of NPs followed the order of iRGD and TAT NPs > TAT-modified NPs > iRGD-modified NPs > primary NPs. iRGD and TAT dual-modified NPs thus displayed significant targeting and penetration ability both in vitro and in vivo, indicating that it is a promising drug delivery system for managing CNV via topical ocular administration.

Increased Active Tumor Targeting by An αvß3-Targeting and Cell-Penetrating Bifunctional Peptide-Mediated Dendrimer-Based Conjugate.

PURPOSE: A bifunctional RGDTAT peptide-modified PEG-PAMAM dendrimer conjugate RGDTAT-PEG-PAMAM (RTPP) was established for the targeted treatment of αvß3-overexpressing tumor cells. METHODS: The RGDTAT peptide was synthesized and attached to PAMAM using PEG to construct the RTPP conjugate. The methotrexate (MTX) encapsulated RTPPM complex was prepared and characterized by dynamic light scattering (DLS), transmission electron microscopy (TEM) and in vitro release. The targeting ability was then studied in cells and tumor-bearing nude mice using fluorescence microscopy, confocal fluorescence microscopy, flow cytometry, and in vivo imaging. The cytotoxicity and pharmacokinetics of the RTPPM complex was also evaluated in cells and rats. RESULTS: The successful synthesis of the RTPP conjugate was confirmed by 1H-NMR. DLS and TEM measurements revealed that the size was 37 nm and the complex had a spherical shape. RTPP and RTPPM were taken up by αvß3-overexpressing cells more efficiently than by αvß3-lowexpressing cells. The RTPP conjugate localized to the cell nucleus and accumulated in the tumor more efficiently than did the conjugates without RGDTAT. The pharmacokinetic study of the RTPPM complex showed sustained drug release. CONCLUSIONS: The bifunctional peptide-mediated dendrimer-based RTPP conjugate can serve as a promising nanocarrier for targeted drug delivery to improve anti-tumor activity.

Intranasal delivery of rotigotine to the brain with lactoferrin-modified PEG-PLGA nanoparticles for Parkinson's disease treatment.

Sustainable and safe delivery of brain-targeted drugs is highly important for successful therapy in Parkinson's disease (PD). This study was designed to formulate biodegradable poly(ethylene glycol)-poly(lactic-co-glycolic acid) (PEG-PLGA) nanoparticles (NPs), which were surface-modified with lactoferrin (Lf), for efficient intranasal delivery of rotigotine to the brain for the treatment of PD. Rotigotine NPs were prepared by nanoprecipitation, and the effect of various independent process variables on the resulting properties of NPs was investigated by a Box-Behnken experimental design. The physicochemical and pharmaceutical properties of the NPs and Lf-NPs were characterized, and the release kinetics suggested that both NPs and Lf-NPs provided continuous, slow release of rotigotine for 48 h. Neither rotigotine NPs nor Lf-NPs reduced the viability of 16HBE and SH-SY5Y cells; in contrast, free rotigotine was cytotoxic. Qualitative and quantitative cellular uptake studies demonstrated that accumulation of Lf-NPs was greater than that of NPs in 16HBE and SH-SY5Y cells. Following intranasal administration, brain delivery of rotigotine was much more effective with Lf-NPs than with NPs. The brain distribution of rotigotine was heterogeneous, with a higher concentration in the striatum, the primary region affected in PD. This strongly suggested that Lf-NPs enable the targeted delivery of rotigotine for the treatment of PD. Taken together, these results demonstrated that Lf-NPs have potential as a carrier for nose-to-brain delivery of rotigotine for the treatment of PD.

Magnetically and pH dual responsive dendrosomes for tumor accumulation enhanced folate-targeted hybrid drug delivery.

Dendrosomes are new tumor targeted drug delivery systems to improve safety and therapeutic effects of antitumor agents. In this study we designed and synthesized magnetically and pH dual responsive dendrosomes with magnetic nanoparticles and folate-targeted dendrimers encapsulated in long-circulating pH sensitive liposomes. Cellular uptake and tissue penetration were assessed on cell lines and tumor spheroids respectively. Xenograft mice were used to study tumor accumulation. The dendrosomes were stable at pH7.4, but responsively released their content at acidic pH. In slightly acid environments, the hybrid vectors showed similar cytotoxicity and cellular uptake to the free folate-dendrimers conjugate due to rapid release. The dendrosomes showed a greater cellular uptake by HeLa cells (FA receptor positive) due to the conjugation with folic acid (FA). In multicellular tumor spheroid tests, a slightly acidic environment and the application of magnet both promoted the permeation efficiency of the hybrid vectors. In the xenograft mice model both in vivo images and tissue distribution assessment indicated that the dendrosomes had higher peak intensity and a longer residence time. Through the synergistic effects of magnetic responsiveness and both passive and active targeting properties, the multi-functional dendrosomes were demonstrated to have great potential as a promising anticancer drug delivery platform.

Synthesis of a bi-functional dendrimer-based nanovehicle co-modified with RGDyC and TAT peptides for neovascular targeting and penetration.

The dual-ligand dendritic polyamidoamine-(polyethylene glycol)n-cyclic RGDyC peptide-(TAT peptide) (PPnR(T)) with various supplied molar ratios of polyethylene glycol (PEG) to polyamidoamine (PAMAM) (n=5, 15, 30) were designed as drug-carriers for the treatment of neovascular diseases; their targeting and penetrating effects were subsequently evaluated. (1)H NMR demonstrated PPnR(T) was successfully synthesized. Compared with the unmodified PAMAM, in vitro cytotoxicity of PPnR(T) to αvß3 negative cells (αvß3-) was significantly reduced, whereas the lethality to pathologic neovascular endothelial cells (αvß3+) was efficiently increased compared to PPn. Compared to PP5R(T) and PP15R(T), PP30R(T) exhibited the most selective and efficient cellular uptake by human umbilical vein endothelial cells (HUVECs, αvß3+). Membrane interaction study indicated the cellular uptake process of PP30R(T) of HUVECs mainly involved specific RGD-αvß3 recognition as well as electrostatic interactions. Intracellular localization results confirmed PP30R(T) was distributed in the cytoplasm in HUVECs. 3D tumor spheroids penetration studies demonstrated that PP30R(T) penetrated the A549 cells to reach the depths of the avascular tumor spheroids. In vivo imaging further demonstrated that PP30R(T) achieved profoundly improved distribution in tumor tissues where angiogenesis existed. Therefore, the bi-functional dendrimer PP30R(T) displayed great potential as a nano-carrier for targeted drug delivery both in vitro and in vivo, and had broad prospects as nanocarriers for the targeted treatment of neovascular diseases.

Preparation, characterization, in vivo pharmacokinetics, and biodistribution of polymeric micellar dimethoxycurcumin for tumor targeting.

Dimethoxycurcumin (DMC) is an analog of curcumin with superior efficacy in various disease models. Currently, drug delivery system research on DMC is very limited, and it has become a huge challenge to realize further developments and clinical applications. In the present study, a kind of amphiphilic block copolymer, N-t-butoxycarbonyl-phenylalanine terminated monomethoxyl poly (ethylene glycol)-b-poly (ε-caprolactone), or mPEG-PCL-Phe(Boc), was prepared from monomethoxyl poly (ethylene glycol)-b-poly (ε-caprolactone) (mPEG-PCL) with its hydroxyl terminal chemically converted into N-t-butoxycarbonyl-phenylalanine (Boc-Phe). This copolymer was determined to have a fairly low critical micelle concentration (2.56×10(-3) mg/mL) and passive targeting potential to tumor tissue, and thus was applied to develop a polymeric micellar formulation of DMC for the first time. The DMC-loaded micelles prepared by thin-film hydration method had typical shell-core structure, with an average particle size of 17.9±0.4 nm and a polydispersity index of 0.045±0.011. The drug loading capacity and entrapment efficiency were 9.94%±0.15% and 97.22%±0.18%, respectively, indicating a high-affinity interaction between DMC and the copolymer. At a concentration of 2 mg/mL, the reconstituted micelle solution could be maintained for at least 10 days at room temperature, and displayed a low initial burst release followed by a sustained release in vitro. Pharmacokinetic study in rats revealed that in vivo drug exposure of DMC was significantly increased and prolonged by intravenously administering DMC-loaded micelles when compared with the same dose of free DMC dissolved in dimethyl sulfoxide. Furthermore, in vivo distribution results from tumor-bearing nude mice demonstrated that this micellar formulation significantly changed the biodistribution profile of DMC and increased drug accumulation in tumors. Therefore, the polymeric micellar formulation of DMC, based on the amphiphilic block copolymer, mPEG-PCL-Phe(Boc), could provide a desirable method for delivering DMC, especially for applications in cancer therapy.