Assessment of alkoxylphenacyl-based polycarbonates as a potential platform for controlled delivery of a model anti-glaucoma drug.

Treatment strategies for glaucoma will benefit from injectable and/or implantable delivery systems that can achieve sustained delivery of neuroprotective agents (to the posterior segment) and/or intraocular pressure lowering drugs (to the anterior segment). In this regard, we have evaluated the suitability of a new polymer (alkoxylphenacyl-based polycarbonates copolymer with polycaprolactone; AP-PCL 20% w/w) as a platform for ocular drug delivery. Brimonidine tartrate (BRT) was applied as a model anti-glaucoma drug. The polymer was applied to develop injectable (nanoparticles) and implantable (microfilms) delivery systems. Nanoparticles fabricated from AP-PCL were stable and have an average size less than 200nm. The AP-PCL microfilms prepared by compression molding showed a gradual hydrolytic in-vitro degradation monitored by water uptake, weight loss, microscopy, DSC and FT-IR measurements. AP-PCL microfilms achieve sustained delivery of BRT for up to 90days. Biocompatibility of AP-PCL-based delivery systems was demonstrated from studies in human trabecular meshwork cell line as well as after intravitreal injections in rats. The overall trend demonstrated that AP-PCL delivery systems may be considered as suitable candidates for prolonged drug delivery in chronic ocular disorders such as glaucoma.

Osteoactivin (GPNMB) ectodomain protein promotes growth and invasive behavior of human lung cancer cells.

The potential application of GPNMB/OA as a therapeutic target for lung cancer will require a greater understanding of the impact of GPNMB/OA ectodomain (ECD) protein shedding into tumor tissues. Thus, in this work we characterized GPNMB/OA expression and extent of shedding of its ECD protein while evaluating the impact on lung cancer progression using three non-small cell lung cancer (NSCLC) cell lines: A549, SK-MES-1 and calu-6. We observed a direct correlation (R2 = 0.89) between GPNMB/OA expression on NSCLC cells and the extent of GPNMB/OA ECD protein shedding. Meanwhile, siRNA-mediated knockdown of GPNMB/OA in cancer cells significantly reduced GPNMB/OA ECD protein shedding, migration, invasion and adhesion to extracellular matrix materials. Also, exogenous treatment of cancer cells (expressing low GPNMB/OA) with recombinant GPNMB/OA protein (rOA) significantly facilitated cell invasion and migration, but the effects of rOA was negated by inclusion of a selective RGD peptide. Further studies in athymic (nu/nu) mice-bearing calu-6 showed that intratumoral supplementation with rOA effectively facilitated in vivo tumor growth as characterized by a high number of proliferating cells (Ki67 staining) coupled with a low number of apoptotic cells. Taken together, our results accentuate the relevance of GPNMB/OA ECD protein shedding to progression of lung cancer. Thus, strategies that suppress GPNMB/OA expression on lung cancer cells as well as negate shedding of GPNMB/OA ECD protein are worthy of consideration in lung cancer therapeutics.

Gelucire-stabilized nanoparticles as a potential DNA delivery system.

Clinical viability of gene delivery systems has been greatly impacted by potential toxicity of the delivery systems. Recently, we reported the nanoparticle (NP) preparation process that employs biocompatible materials such as Gelucire® 44/14 and cetyl alcohol as matrix materials. In the current study, the NP preparation was modified for pDNA loading through: (i) inclusion of cationic lipids (DOTAP or DDAB) with NP matrix materials; or (ii) application of cationic surfactants (CTAB) to generate NPs with desired surface charges for pDNA complexation. Colloidal stability and efficiency of loading pGL3-DR4X2-luciferase plasmid DNA in NPs were verified by gel permeation chromatography. Compared to pDNA alone, all the NPs were effective in preserving pDNA from digestion by DNase. While pDNA loading using CTAB-NPs involved fewer steps compared to DOTAP-NPs and DDAB-NPs, CTAB-NPs were greatly impacted by elevated cytotoxicity level which could be ascribed to the concentrations of CTAB in NP formulations. In vitro transfection studies (in HepG2 cells) based on luciferase expression showed the ranking of cell transfection efficiency as DOTAP-NPs > DDAB-NPs > CTAB-NPs. The overall work provided an initial assessment of gelucire-stabilized NPs as a potential platform for gene delivery.

Brain-targeted delivery of doxorubicin using glutathione-coated nanoparticles for brain cancers.

OBJECTIVES: To prepare and characterize in vitro a novel brain-targeted delivery of doxorubicin using glutathione-coated nanoparticles (NPs) for the treatment of brain cancer. METHODS: Doxorubicin-loaded NPs were prepared by the nanoprecipitation method using PLGA-COOH (dl-lactide-co-glycolide). The NPs were coated with a glutathione-PEG conjugate (PEG-GSH) in order to target delivery to the brain. The NPs were characterized via in vitro studies to determine particle size, drug release, cellular uptake, immunofluorescence study, cytotoxic assay, and in vitro blood-brain barrier (BBB) assay. RESULTS: The NPs showed a particle size suitable for BBB permeation (particle size around 200 nm). The in vitro release profile of the NPs exhibited no initial burst release and showed sustained drug release for up to 96 h. The immunofluorescence study showed the glutathione coating does not interfere with the drug release. Furthermore, in vitro BBB Transwell™ study showed significantly higher permeation of the doxorubicin-loaded NPs compared with the free doxorubicin solution through the coculture of rat brain endothelial (RBE4) and C6 astrocytoma cells (p < 0.05). CONCLUSIONS: We conclude that the initial in vitro characterization of the NPs demonstrates potential in delivering doxorubicin to cancer cells with possible future application in targeting brain cancers in vivo.

Emerging lung cancer therapeutic targets based on the pathogenesis of bone metastases.

Lung cancer is the second most common cancer and the leading cause of cancer related mortality in both men and women. Each year, more people die of lung cancer than of colon, breast, and prostate cancers combined. It is widely accepted that tumor metastasis is a formidable barrier to effective treatment of lung cancer. The bone is one of the frequent metastatic sites for lung cancer occurring in a large number of patients. Bone metastases can cause a wide range of symptoms that could impair quality of life of lung cancer patients and shorten their survival. We strongly believe that molecular targets (tumor-related and bone microenvironment based) that have been implicated in lung cancer bone metastases hold great promise in lung cancer therapeutics. Thus, this paper discusses some of the emerging molecular targets that have provided insights into the cascade of metastases in lung cancer with the focus on bone invasion. It is anticipated that the information gathered might be useful in future efforts of optimizing lung cancer treatment strategies.

Formulation and photoirradiation parameters that influenced photoresponsive drug delivery using alkoxylphenacyl-based polycarbonates.

Recently, we reported the synthesis and biocompatibility of alkoxylphenacyl-based polycarbonates (APP); a promising new class of polymers that undergo photo-induced chain scission. In the current study, nanoparticles (NPs) were prepared from the APP polymer (APP-NPs) and loaded with doxorubicin (DOX) (DOX-APP-NPs) in order to identify and evaluate formulation and photoirradiation parameters that influence photoresponsive efficacy. Stable and spherical APP-NPs were prepared with diameters between 70-80nm depending on APP concentration (10-40mg/mL). There was a direct relationship between APP concentration and resultant particle size. Drug release studies indicated that exposure to the photo-trigger was capable of altering the rate and extent of DOX released. Photoresponsive DOX release was markedly influenced by the frequency of photoirradiation while the effect of APP concentration was most likely propagated through NP size. DOX released by photoactivation retained its efficacy as assessed by cytotoxicity studies in human lung adenocarcinoma (A549) cells. Studies in BALB/c mice indicated that DOX-APP-NPs induce less cardiotoxicity than DOX alone and that DOX-APP-NPs are not susceptible to dose dumping after photoirradiation.

Antitumor efficacy and tolerability of systemically administered gallium acetylacetonate-loaded gelucire-stabilized nanoparticles.

The widespread clinical success with most gallium compounds in cancer therapy is markedly hampered by lack of tumor specific accumulation, poor tumor permeability and undesirable toxicity to healthy tissues. The aim of this work was to investigate for the first time antitumor mechanism of a new gallium compound (gallium acetylacetonate; GaAcAc) while assessing effectiveness of gelucire-stabilized nanoparticles (NPs) for potential application in gallium-based lung cancer therapy. NPs loaded with GaAcAc (Ga-NPs) were prepared using mixtures of cetyl alcohol with Gelucire 44/14 (Ga-NP-1) or Gelucire 53/13 (Ga-NP-2) as matrix materials. Of special note from this work is the direct evidence of involvement of microtubule disruption in antitumor effects of GaAcAc on human lung adenocarcinoma (A549). In-vivo tolerability studies were based on plasma ALT, creatinine levels and histopathological examination of tissues. The superior in-vivo antitumor efficacy of Ga-NPs over GaAcAc was depicted in marked reduction of tumor weight and tumor volume as well as histological assessment of excised tumors. Compared to free GaAcAc, Ga-NPs showed a 3-fold increase in tumor-to-blood gallium concentrations with minimized overall exposure to healthy tissues. Overall, enhancement of antitumor effects of GaAcAc by gelucire-stabilized NPs coupled with reduced exposure of healthy tissues to gallium would likely ensure desired therapeutic outcomes and safety of gallium-based cancer treatment.

Biocompatibility and in vivo tolerability of a new class of photoresponsive alkoxylphenacyl-based polycarbonates.

Potential toxicities of chromophoric or polymeric units of most photoresponsive delivery systems have impacted clinical relevance. Herein, we evaluated the biocompatibility and tolerability of alkoxylphenacyl-based polycarbonates (APPs) as a new class of photoresponsive polymers. The polymers were applied as homopolymer or copolymers of polyethylene glycol (10%, w/w) or polycaprolactone (10%, w/w). APP polymers were comparable to poly(lactic-co-glycolic acid) (PLGA) based on cytotoxicity, macrophage activation, and blood compatibility. Data from biodistribution studies in BALB/c mice showed preferential accumulation in kidney and liver. Meanwhile, potential application of APP polymers as immediate or sustained (implants) drug delivery systems indicated that liver and kidney functions were not distorted. Also, plasma levels of tumor necrosis factor-alpha and interleukin-6 were comparable to PLGA-treated mice (p > 0.05). A histological analysis of liver and kidney sections showed no detectable damage for APP polymers. The overall data strongly supported potential consideration of APP polymers as photoresponsive delivery systems especially as implantable or tissue-mimicking photopatterned biomaterials.

Thermoreversible gel for delivery of activin receptor-like kinase 5 inhibitor SB-505124 for glaucoma filtration surgery.

The purpose of this study is to investigate a thermoreversible gel using Pluronic F-127 to deliver an activin receptor-like kinase 5 (ALK-5) inhibitor SB-505124 in glaucoma filtration surgery (GFS). The gel was characterized for in vitro drug release and viscosity studies. Cytotoxicity of Pluronic F-127 was examined by MTT assay using cultured rabbit subconjunctival fibroblasts. In addition, Pluronic F-127 gel (18% w/v) containing 5 mg of SB-505124 was applied at the surgical site in an in vivo rabbit GFS model. In the in vitro viscosity study, the gel showed a change in viscosity (from 1000 cps to 45,000 cps) from low temperature (10°C) to body temperature (37°C). The in vitro drug release study demonstrated 100% drug release within 12 h. The gel did not show cytotoxicity to the cultured rabbit subconjunctival cells by MTT assay. In the in vivo rabbit GFS model, the drug was successfully delivered by injection and no severe post-surgical complications were observed. A thermoreversible gel system with SB-505124 was successfully prepared and delivered for the rabbit GFS model, and it may provide a novel delivery system in GFS.

Contributory roles of innate properties of cetyl alcohol/gelucire nanoparticles to antioxidant and anti-inflammation activities of quercetin.

The protective effects of synthetic lung surfactant Exosurf® (containing cetyl alcohol) against endotoxin-induced inflammation have been demonstrated in the literature. Thus, it is envisioned that nanoparticles loaded with quercetin (Q-NPs) prepared with binary mixtures of cetyl alcohol (CA) and Gelucire 44/14® (gelucire) as matrix materials will be capable of overcoming some of the protracted challenges confronting clinical application of quercetin and possess innate protective activity against inflammatory responses, which could be synergistic with quercetin. The NPs were stable in simulated biological media while retaining their particle size and spherical morphology. Further analysis by gel permeation chromatography, spectroscopic analysis (ultraviolet-visible, fluorescence, and Fourier transform infrared spectroscopy) indicated entrapment of quercetin in NPs. Q-NPs effectively enhanced xanthine oxidase inhibitory and free radical scavenging effect of quercetin. Furthermore, Q-NPs showed marked reduction (compared to quercetin alone) in production of nitric oxide and cytokine (interleukin-6 and tumor necrosis factor alpha) from lipopolysaccharide-activated macrophages. Superiority of Q-NPs over quercetin alone was confirmed from in vivo anti-inflammatory efficacy studies in BALB/c mice. Data from additional studies with blank NPs (without quercetin) showed that the NPs reported herein most likely possessed intrinsic protective properties against LPS-induced inflammation. Although further mechanistic studies are warranted, the overall work depicted a novel approach of possible exploiting innate protective properties of NPs in quercetin delivery for treating oxidative stress and inflammation.

Biocompatibility, efficacy and biodistribution of Gelucire-stabilized nanoparticles engineered for docetaxel delivery.

Docetaxel is a potent anticancer agent that will benefit greatly from alternative delivery systems that can overcome several reported adverse effects due to the drug itself and/or the solvent system in the current clinical formulation. In this regard, a new nanoparticle delivery system for docetaxel was prepared from Gelucire-based nanoemulsions by using binary mixtures of Gelucire 44/14 and cetyl alcohol as NP matrix materials. Various amounts of docetaxel (50-1000 microg/ml) were added to the oil phase of the nanoemulsions prior to obtaining solid nanoparticles. The nanoparticles (100-140 nm) achieved high entrapment efficiency (> or = 89%) of docetaxel which was maintained upon storage at 4 degrees C and 25 degrees C. Additional data indicated the Gelucire component in NP played influential roles in drug release possibly by facilitating diffusion from NPs and/or accelerating erosion of NP matrix. Docetaxel-loaded nanoparticles did not cause any significant red blood cell lysis or platelet aggregation nor activate macrophages. Also in-vitro antitumor efficacy in human lung adenocarcinoma cells was demonstrated based on cell cytotoxicity, production of reactive oxygen species and reduction of mitochondrial potential. Enhancement of in-vitro antitumor effects of docetaxel with Gelucire-based NPs could be ascribed to improved particle dispersion and efficient cell permeability. Studies in BALB/c mice demonstrated the stability/retention of NPs in blood circulation and the potential in facilitating docetaxel absorption across the peritoneal cavity. The nanoparticles reported herein may be effective as novel biocompatible and effective delivery systems for docetaxel.

Antitumor effect of novel gallium compounds and efficacy of nanoparticle-mediated gallium delivery in lung cancer.

The widespread application of gallium (Ga) in cancer therapy has been greatly hampered by lack of specificity resulting in poor tumor accumulation and retention. To address the challenge, two lipophilic gallium (III) compounds (gallium hexanedione; GaH and gallium acetylacetonate; GaAcAc) were synthesized and antitumor studies were conducted in human lung adenocarcinoma (A549) cells. Nanoparticles (NPs) containing various concentrations of the Ga compounds were prepared using a binary mixture of Gelucire 44/14 and cetyl alcohol as matrix materials. NPs were characterized based on size, morphology, stability and biocompatibility. Antitumor effects of free or NP-loaded Ga compounds were investigated based on cell viability, production of reactive oxygen species and reduction of mitochondrial potential. Compared to free Ga compounds, cytotoxicity of NP-loaded Ga (5-150 microg/ml) was less dependent on concentration and incubation time (exposure) with A549 cells. NP-mediated delivery (5-150 microg Ga/ml) enhanced antitumor effects of Ga compounds and the effect was pronounced at: (i) shorter incubation times; and (ii) at low concentrations of gallium (approximately 50 microg/ml) (p < 0.0006). Additional studies showed that NP-mediated Ga delivery was not dependent on transferrin receptor uptake mechanism (p > 0.13) suggesting the potential in overcoming gallium resistance in some tumors. In general, preparation of stable and biocompatible NPs that facilitated Ga tumor uptake and antitumor effects could be effective in gallium-based cancer therapy.

Effects of gelucire content on stability, macrophage interaction and blood circulation of nanoparticles engineered from nanoemulsions.

The main objective of the study is to investigate the efficacy of Gelucire 44/14 (gelucire) in facilitating formation of cetyl alcohol (CA)-based nanoparticle (NP) and to assess the effects on key NP properties and functions. NPs from oil-in-water nanoemulsion precursors were prepared using binary mixtures of CA and gelucire (CA/gelucire) containing gelucire at 0, 25, 50 and 75% (w/w). The sizes of gelucire-based NPs (128-183 nm) were five times lower than control NPs (made without gelucire). All the NPs (with or without gelucire component) did not activate macrophages as monitored by reactive oxygen species production. Results from differential scanning calorimetry, FT-IR and multimodal light scattering measurements demonstrated the involvement of gelucire component in achieving homogeneous CA/gelucire particle populations that were stable on storage. The P-glycoprotein (P-gp) function assay in MES-Dx5 cells showed the potential of gelucire-based NPs in inhibiting rhodamine 123 efflux. Similarly, the extent of NP uptake by macrophage (RAW 264.7 cell) was dependent on the amount of gelucire component (inverse relationship; R(2)=0.996). NPs made with CA/gelucire mixture (at 50%, w/w gelucire) were the most effective in blood circulation studies in BALB/c mice. Additional studies with paclitaxel-loaded NPs demonstrated that the retention of gelucire-based NPs in blood circulation was comparable to NPs coated with DSPE-PEG(2000) (p>0.6). The over-all work indicated the potential efficacy of gelucire as a safe and biocompatible excipient that can serve multiple functions in enhancing the performance of lipid-based NP drug delivery systems.

Development and validation of a novel RP-HPLC method for the analysis of reduced glutathione.

The objective of this study was the development, optimization, and validation of a novel reverse-phase high-pressure liquid chromatography (RP-HPLC) method for the quantification of reduced glutathione in pharmaceutical formulations utilizing simple UV detection. The separation utilized a C18 column at room temperature and UV absorption was measured at 215 nm. The mobile phase was an isocratic flow of a 50/50 (v/v) mixture of water (pH 7.0) and acetonitrile flowing at 1.0 mL/min. Validation of the method assessed the methods ability in seven categories: linearity, range, limit of detection, limit of quantification, accuracy, precision, and selectivity. Analysis of the system suitability showed acceptable levels of suitability in all categories. Likewise, the method displayed an acceptable degree of linearity (r(2) = 0.9994) over a concentration range of 2.5-60 µg/mL. The detection limit and quantification limit were 0.6 and 1.8 µg/mL respectively. The percent recovery of the method was 98.80-100.79%. Following validation the method was employed in the determination of glutathione in pharmaceutical formulations in the form of a conjugate and a nanoparticle. The proposed method offers a simple, accurate, and inexpensive way to quantify reduced glutathione.