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The goal of this study was to develop bioadhesive food protein nanoparticles using zein (Z), a hydrophobic corn protein, as the core and whey protein (WP) as the shell for oral pediatric drug delivery applications. Lopinavir (LPV), an antiretroviral drug, and fenretinide, an investigational anticancer agent, were used as model drugs in the study. The particle size of ZWP nanoparticles was in the range of 200-250 nm, and the drug encapsulation efficiency was >70%. The nanoparticles showed sustained drug release in simulated gastrointestinal fluids. ZWP nanoparticles enhanced the permeability of LPV and fenretinide across Caco-2 cell monolayers. In both ex vivo and in vivo studies, ZWP nanoparticles were found to be strongly bioadhesive. ZWP nanoparticles enhanced the oral bioavailability of LPV and fenretinide by 4 and 7-fold, respectively. ZWP nanoparticles also significantly increased the half-life of both drugs. The nanoparticles did not show any immunogenicity in mice. Overall, the study demonstrates the feasibility of developing safe and effective food protein-based nanoparticles for pediatric oral drug delivery.

Almost all breast cancers originate from epithelial cells lining the milk ducts in the breast. To this end, the study investigated the feasibility of localized transdermal delivery of α-santalol, a natural chemopreventive agent to the breast. Different α-santalol formulations (cream, solution and microemulsion) were developed and the in vitro permeability was studied using excised animal (porcine and rat) and human breast skin/mammary papilla (nipple). The in vivo biodistribution and efficacy studies were conducted in female rats. A chemical carcinogenesis model of breast cancer was used for the efficacy studies. Phospholipid based α-santalol microemulsion showed the highest penetration through the nipple and breast skin. Delivery of α-santalol through the entire breast (breast skin and nipple) in vivo in rats resulted in significantly higher concentration in the mammary gland compared to transdermal delivery through the breast skin or nipple. There was no measurable α-santalol concentration in the blood. Transdermal delivery of α-santalol reduced the tumor incidence and tumor multiplicity. Furthermore, the tumor size was significantly reduced with α-santalol treatment. The findings from this study demonstrate the feasibility of localized transdermal delivery of α-santalol for chemoprevention of breast cancer.

Drug delivery by direct intraductal administration can achieve high local drug concentration in the breast and minimize systemic levels. However, the clinical application of this approach for breast cancer treatment is limited by the rapid clearance of the drug from the ducts. With the goal of developing strategies to prolong drug retention in the breast, this study was focused on understanding the influence of particle size and formulation on breast duct and lymph node retention. Fluorescent-labeled polystyrene (PS) particles ranging in size from 100 to 1000 nm were used to study the influence of particle size. Polylactic acid-co-glycolic acid (PLGA) was used to develop and test formulations for intraductal delivery. Cy 5.5, a near-IR dye, was encapsulated in PLGA microparticles, nanoparticles, and the in situ gel to study the biodistribution in rats using an in vivo imager. PS microparticles (1 μm) showed longer retention in the duct compared to 100 and 500 nm nanoparticles. The ductal retention half-life was 5-fold higher for PS microparticles compared to the nanoparticles. On the other hand, the free dye was cleared from the breast within 6 h. PLGA nanoparticles sustained the release of Cy 5.5 for >4 days. Microparticles and gel showed a much slower release than nanoparticles. PLGA in situ gel and microparticles were retained in the breast for up to 4 days, while the nanoparticles were retained in the breast for 2 days. PLGA nanoparticles and microparticles drained to the axillary lymph node and were retained for up to 24 and 48 h, respectively, while the in situ gel and the free dye did not show any detectable fluorescence in the lymph nodes. Taken together, the results demonstrate the feasibility of prolonged retention in the breast duct and lymph node by optimal formulation design. The findings can serve as a framework to design formulations for localized treatment of breast cancer.

The study was aimed at systematically investigating the influence of shell composition on the particle size, stability, release, cell uptake, permeability, and in vivo gastrointestinal distribution of food protein based nanocarriers for oral delivery applications. Three different core-shell nanocarriers were prepared using food-grade biopolymers including zein-casein (ZC) nanoparticles, zein-lactoferrin (ZLF), nanoparticles and zein-PEG (ZPEG) micelles. Nile red was used as a model hydrophobic dye for in vitro studies. The nanocarriers had negative, positive, and neutral charge, respectively. All three nanocarriers had a particle size of less than 200 nm and a low polydispersity index. The nanoparticles were stable at gastrointestinal pH (2-9) and ionic strength (10-200 mM). The nanocarriers sustained the release of Nile red in simulated gastric and intestinal fluids. ZC nanoparticles showed the slowest release followed by ZLF nanoparticles and ZPEG micelles. The nanocarriers were taken up by endocytosis in Caco-2 cells. ZPEG micelles showed the highest cell uptake and transepithelial permeability followed by ZLF and ZC nanoparticles. ZPEG micelles also showed P-gp inhibitory activity. All three nanocarriers showed bioadhesive properties. Cy 5.5, a near IR dye, was used to study the in vivo biodistribution of the nanocarriers. The nanocarriers showed longer retention in the rat gastrointestinal tract compared to the free dye. Among the three formulations, ZC nanoparticles was retained the longest in the rat gastrointestinal tract (≥24 h). Overall, the outcomes from this study demonstrate the structure-function relationship of core-shell protein nanocarriers. The findings from this study can be used to develop food protein based oral drug delivery systems with specific functional attributes.

Glutathione disulfide (GSSG) is the oxidized form of glutathione (GSH). GSH is a tripeptide present in the biological system in mM concentration and is the major antioxidant in the body. An increase in GSSG reflects an increase in intracellular oxidative stress and is associated with disease sates. The increase has also been demonstrated to lead to an increase in protein S-glutathionylation that can affect the structure and function of proteins. Protein S-glutathionylation serves as a regulatory mechanism during cellular oxidative stress. Though GSSG is commercially available, its roles in various GSSG-associated normal/abnormal physiological functions have not been fully delineated due to the reason that GSSG is not cell membrane permeable and a lack of method to specifically increase GSSG in cells. We have developed cationic liposomes that can effectively deliver GSSG into cells. Various concentrations of GSSG liposomes can be conveniently prepared. At 1 mg/mL, the GSSG liposomes effectively increased intracellular GSSG by 27.1±6.9 folds (n=3) in 4 h and led to a significant increase in protein S-glutathionylation confirming that the increased GSSG is functionally effective. The Trypan blue assay demonstrated that GSSG liposomes were not cytotoxic; the cell viability was greater than 95% after cells were treated with the GSSG liposomes for 4 h. A stability study showed that the dry form of the GSSG liposomes were stable for at least 70 days when stored at −80 °C. Our data demonstrate that the GSSG liposomes can be a valuable tool in studying GSSG-associated physiological/pathological functions., Highlights • Glutathione disulfide (GSSG) liposomes were developed to deliver GSSG into cells. • The GSSG liposomes effectively increased intracellular GSSG by 27.1±6.9 folds. • The delivered GSSG is confirmed to be functionally effective through protein S-glutathionylation.

Novel biodegradable micelles were synthesized by conjugating methoxy poly(ethylene glycol) (mPEG) to zein, a biodegradable hydrophobic plant protein. The mPEG-zein micelles were in the size range of 95-125 nm with a low CMC (5.5 × 10(-2) g/L). The micelles were nonimmunogenic and were stable upon dilution with buffer as well as 10% serum. Curcumin, an anticancer agent with multiple delivery challenges, was encapsulated in mPEG-zein micelles. The micelles significantly enhanced the aqueous solubility (by 1000-2000-fold) and stability (by 6-fold) of curcumin. PEG-zein micelles sustained the release of curcumin up to 24 h in vitro. Curcumin-loaded mPEG-zein micelles showed significantly higher cell uptake than free curcumin in drug-resistant NCI/ADR-RES cancer cells in vitro. Micellar curcumin formulation was more potent than free curcumin in NCI/ADR-RES cancer cells, as evidenced from the 3-fold reduction in IC(50) value of curcumin. Overall, this study for the first time reports a natural protein core based polymeric micelle and demonstrates its application for the delivery of hydrophobic anticancer drugs such as curcumin.

To investigate skin penetration of poly (amidoamine) (PAMAM) dendrimers as a function of surface charge and molecular weight in presence and absence of iontophoresis. Dendrimers were labeled with fluoroisothiocynate (FITC); skin penetration of dendrimers was studied using excised porcine skin in-vitro. Skin penetration of FITC-labeled dendrimers was quantified using confocal laser scanning microscope (CLSM). G2-G6 NH2, G3.5-COOH and G4-OH dendrimers were used. Cationic dendrimers showed higher skin penetration than neutral and anionic dendrimers. Skin penetration of cationic dendrimer increased linearly with increase in treatment time. Iontophoresis enhanced skin penetration of cationic and neutral dendrimers. Increase in current strength and current duration increased skin transport of dendrimers. Passive and iontophoretic skin penetration of cationic dendrimers was inversely related to their molecular weight. Dendrimer penetrated the skin through intercellular lipids and hair follicles. With iontophoresis, dendrimer was also found in localized skin regions. The study demonstrates that the physicochemical properties of dendrimers influence their skin transport. Findings can be used to design dendrimer-based nanocarriers for drug delivery to skin.

The aim of present study is to investigate the effect of poly(amidoamine) (PAMAM) dendrimer on skin permeation of 5-fluorouracil (5FU). Permeation studies were performed using excised porcine skin in a Franz diffusion cell and 14C labeled 5FU samples were analyzed using liquid scintillation counter. Three different vehicles were used, including phosphate buffer (PB), mineral oil (MO) and isopropyl myristate (IPM). The studies were carried out by simultaneously applying the drug and dendrimer together or by pre-treating the skin with dendrimer before drug application. Simultaneous application of drug and dendrimer increased the flux of 5FU in IPM and MO, while there was no change in PB. The increased skin partitioning of dendrimer from lipophilic vehicles increased the drug solubility in skin. Pre-treatment with dendrimer increased permeability coefficient of 5FU by 4-fold in MO and 2.5-fold in IPM, while it decreased by half in PB. Skin partitioning of 5FU increased after dendrimer treatment from lipophilic vehicles. The flux increased linearly with increase in pre-treatment time. Dendrimer pre-treatment increased transepidermal water loss and decreased skin resistance. The decrease in skin resistance directly correlated to the enhancement in skin permeation of 5FU (r2 = 0.99). Overall, the study showed that dendrimer increases the skin permeation of 5FU from lipophilic vehicles mainly by altering the skin barrier.

Skin is an important site for local or systemic application of drugs. However, a majority of drugs have poor permeability through the skin's topmost layer, stratum corneum (SC). The aim of this study was to identify safe and smaller peptides that could enhance the skin penetration of drug molecules. By screening phage display peptide library, we have identified a T2 peptide (LVGVFH), which enhanced the penetration of bacteriophages (~800 nm long bacterial viruses) across porcine and mouse skin. Pretreating the skin with synthetic T2 peptide at pH 4.5 resulted in significant penetration enhancement of hydrophilic drug 5-fluorouracil (5-FU) across skin. FTIR spectroscopy showed that the T2 peptide interacted with skin lipids to enhance the skin penetration. Pretreating the skin with T2 peptide enhanced the partitioning of small molecules with different lipophilicities (5-FU, fluorescein isothiocyanate, and rhodamine 123 hydrochloride) into skin. Fluorescence studies showed that T2 peptide enhanced the diffusion of these molecules into intercellular lipids of SC and thus enhanced the penetration into the skin. Histidine at the c-terminus of T2 peptide was identified to be critical for the skin penetration enhancement. T2 peptide interacted with skin lipids to cause skin penetration enhancement. The study identified a novel, safe, and noninvasive peptide to improve the skin penetration of drugs without chemical conjugation.

Zein is a hydrophobic water insoluble plant protein and the main goal of the present study was to prepare zein nanoparticles using pH controlled nanoprecipitation method. Nanoparticle characteristics such as size, polydispersity index (PI), zeta potential, and encapsulation efficiency were studied using 6, 7-dihydroxycoumarin, as a model hydrophobic compound. Lecithin and pluronic F68 were used as stabilizers. The blank zein nanoparticles had a mean particle size of 460 nm; while coumarin loaded zein nanoparticles had a mean particle size of 365 nm. The encapsulation efficiency of coumarin loaded zein nanoparticles was 62%. The release of coumarin from zein nanoparticles was sustained in phosphate buffer (pH 7.4) for upto 9 days. Overall the results from this study demonstrate a new method for preparing drug loaded zein nanoparticles.

The study investigates the influence of surface charge, generation and concentration of poly(amidoamine) (PAMAM) dendrimers on skin permeation of a model hydrophilic drug, 5-fluorouracil (5FU). Permeation studies were performed using excised porcine skin in a Franz diffusion cell. Saturated concentration of 5FU in isopropyl myristate was applied on the skin after pretreatment with dendrimers and 14 C labeled 5FU was analyzed using a liquid scintillation counter. The influence of dendrimer surface charge (G4-NH 2 , G3.5-COOH, and G4-OH), generations (G2-G6-NH 2 ) and concentration (0.1–10 mM of G4-NH 2 ) on skin permeation of 5FU were studied. The enhancement in drug permeability coefficient ( K p ) was in the following decreasing order G4-NH 2 > G4-OH > G3.5-COOH. Dendrimer increased the skin permeation by increasing the skin partitioning of 5FU. Transepidermal water loss, skin resistance measurements and ATR-FTIR studies revealed that cationic dendrimers act by interacting with the skin lipid bilayers. Increase in G4-NH 2 concentration did not proportionally increase the skin permeation of 5FU. The 5FU K p was inversely related to the molecular weight of the dendrimer. G2-NH 2 dendrimer reduced skin resistance to a greater extent than higher generation dendrimers. Overall, the study showed that lower generation cationic dendrimer is more effective in enhancing the skin permeation of hydrophilic drugs. © 2008 Wiley-Liss, Inc. and the American Pharmacists Association.

Purpose: Honokiol, a plant lignan isolated and purified from Magonila Officinalis has been shown to have chemopreventive effects in chemically induced and UV-B induced skin tumor development in SKH-1 mice. However poor solubility of honokiol greatly limits its clinical application. The key objectives of the present investigation are to improve the solubility and stability of honokiol by encapsulating in nanostructured lipid nanoparticles. Methods: Honokiol was incorporated into nanostructured lipid nanoparticles and cream using hot homogenization method. Compritol 888 ATO, Precirol ATO 5, and miglyol 812 as lipid matrices and poloxamer 188 as emulsifier. Lipid nanoparticles were characterized for size, zeta potential, shape and morphology using photon correlation spectroscopy (PCS) and atomic force microscopy (AFM). Honokiol was quantified by isocratic HPLC method using mobile phase of 60:40 (%v/v, acetonitrile:water) at a flow rate of 1 ml/min at a wavelength of 254 nm. Permeation studies were carried out using porcine dermatome skin in Franz diffusion cell. Honokiol concentration corresponding to 0.5 mg/ml in sesame oil, cream and lipid nanoparticles was dispersed in PBS pH 7.4. Permeation studies were performed till 24hrs; the samples (200 µl) were withdrawn at regular time intervals and replaced with fresh medium. Amount of honokiol permeated across porcine skin was quantified using HPLC method. Stability of cream and lipid nanoparticle formulations was studied at 30° C and 65 % relative humidity in a stability chamber for one month. Results: The size of the optimized honokiol loaded lipid nanoparticles was 145±7 nm with a polydispersity index of 0.24±0.02. The zeta potential and encapsulation efficiency was 0.34±0.02 mV and 97±2 %. The particle size of lipid nanoparticles measured in AFM was very close to size measured by PCS. In case of precirol cream, particle size was more than 20 µm with drug content of 89±5 %. Honokiol loaded nanostructured lipid nanoparticles, and cream were stable for about 30 days without any significant changes in particle size, and drug content at 30° C and 65 % relative humidity. Skin treated with honokiol entrapped in nanoparticles and cream has shown more retention of honokiol in the skin compared to plain honokiol in sesame oil. Proposed lipid cream and nanoparticles formulation has shown effective skin penetration of honkiol. From the data it is evident that nanoparticles has shown higher amount of honokiol in skin compared to cream formulation. Conclusions: Preliminary results of this study have shown the potential of lipid based formulations for topical delivery of honokiol. Chemopreventive potential and efficacy of honokiol encapsulated in lipid based formulations will be evaluated in UV-B induced skin cancer model. Acknowledgements: Supported by Translational Cancer Research Center funded by the South Dakota Governor's Office of Economic Development. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5709. doi:1538-7445.AM2012-5709

Skin cancer is the most prevalent of all cancers and its incidence is expected to increase substantially. UVB is major etiologic factor in the skin cancer development. Honokiol, a plant ligan isolated from bark and seed cones of Magnolia officinalis, has been shown to have chemopreventive effects on UVB-induced skin cancer development in our laboratory. The objectives of this investigation are to study chemopreventive effects of dose response of honokiol on UVB-induced skin tumor development in SKH-1mice and to prepare topical formulations of honokiol that deliver effective concentration of drug locally. Female SKH-1 mice were divided into 4 groups. Group 1 received acetone one hour before UVB treatment; Group 2, 3 and 4 received honokiol (0.2 ml of 30, 45 and 60 μg in acetone, topical) before each UVB treatment. Mice were treated with UVB dose of 30 mJ/cm2/day for 25 weeks. Tumor counts and group weights were taken weekly. Honokiol pretreated groups resulted in 36-78% reduction in tumor multiplicity compared to control. Honokiol pretreatment (60 μg) decreased tumor incidence by 40 %. Topical formulation of honokiol gel was prepared by using carbomer 940 as base and propylene glycol as penetration enhancer. Drug content and content uniformity were determined. Release studies and permeation studies were studied in detail. This study is supported by Translational Cancer Research Center funded as 2010 Research Initiative Center by the State of South Dakota Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4620. doi:10.1158/1538-7445.AM2011-4620