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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.

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.

The study was aimed at investigating the feasibility of using a poly (amidoamine) (PAMAM) dendrimer as a carrier for topical iontophoretic delivery of an antisense oligonucleotide (ASO). Bcl-2, an anti-apoptotic protein implicated in skin cancer, was used as the model target protein to demonstrate the topical gene silencing approach. Confocal laser scanning microscopy studies demonstrated that the iontophoretically delivered ASO-dendrimer complex can reach the viable epidermis in porcine skin. In contrast, passively delivered free or dendrimer complexed ASO was mainly localized to the stratum corneum. The cell uptake of ASO was significantly enhanced by the dendrimer complex and the complex suppressed Bcl-2 levels in the cell. In the skin cancer mouse model, the iontophoretically delivered ASO-dendrimer complex reduced the tumor volume by 45% and was consistent with the reduction in Bcl-2 protein levels. The iontophoretically delivered ASO-dendrimer complex caused significant apoptosis in skin tumor. Overall, the findings from this study demonstrate that dendrimers are promising nanocarriers for developing topical gene silencing approaches for skin diseases.

The main objective of this study was to investigate the potential of calcium phosphate (CAP) nanoparticles for transcutaneous vaccine delivery. CAP nanoparticles were prepared by nanoprecipitation method followed by sequential adsorption of sugars and ovalbumin. Nanoparticles were characterized using dynamic light scattering, XRD, ATR-FTIR, and microscopy methods. In-vitro release of ovalbumin from nanoparticles was studied in phosphate buffer (pH 7.4). In-vivo immunization studies were carried out in Balb/C mice. The size and zeta potential of ovalbumin-sugar adsorbed CAP nanoparticles was 350 +/- 22.5 nm and -12.93 +/- 1.02 mV respectively. Around 60% ovalbumin was released from nanoparticles within 24 hrs. To test the feasibility for transcutaneous vaccine delivery, the nanoparticles were applied in mice after removing the stratum corneum by tape-stripping. In the positive control group, the nanoparticles were administered by intradermal injection. Ovalbumin-sugar coated CAP nanoparticles showed significantly higher antibody titers (Total IgG and IgG1) compared to ovalbumin alone. IgG2a antibodies were only seen with intradermal injection. Both topical and intradermal treatment groups showed splenocyte proliferation when re-stimulated with ovalbumin. The results from this study demonstrate the potential of using CAP nanoparticles for transcutaneous vaccine delivery.

Majority of breast cancers originate from epithelial cells in the duct and lobules in the breast. Current systemic treatments for breast cancer are associated with significant systemic side effects, thus warranting localized drug delivery approaches. The aim of this study was to investigate the influence of hydroalcoholic vehicle on topical delivery of 4-hydroxy tamoxifen (4-HT) through the mammary papilla (nipple). The in vitro permeability of 4-HT through porcine mammary papilla was studied using different hydroalcoholic vehicles (0, 33.33, and 66.66% alcohol). Nile red was used as a model lipophilic dye to characterize the drug transport pathway in the mammary papilla. The penetration of 4-HT through the mammary papilla increased with increase in alcohol concentration in the vehicle. The solubility of 4-HT was enhanced by increasing alcohol concentration in the vehicle. On the other hand, the epidermis/vehicle partition coefficient decreased with increase in alcohol concentration. The mammary papilla served as a depot and slowly released 4-HT into the receptor medium. Highest drug penetration was observed with saturated drug solution in 66.66% alcohol, and 4-HT levels were comparable to IC50 value of 4-HT. Results from this study demonstrate the possibility of using mammary papilla as a potential route for direct delivery of 4-HT to the breast.

INTRODUCTION Skin is the largest organ in the human body and functions as a protective barrier. The large surface area (1.8 m2) and easy accessibility of skin make it one of the attractive routes for drug delivery. However, the unique bioarchitecture of skin limits the transport of molecules through it (1). The skin is primarily divided into epidermis, dermis, and subcutaneous tissue. Epidermis is again subdivided into stratum corneum (SC), stratum lucidum, stratum granulosum, stratum germinativum, and stratum basale [Fig. 1(A)]. Stratum basale is the germinal layer which continuously divides to produce new keratinocytes that move to outer layers finally to form a horny layer of dead cells in the SC. It takes 2 to 3 weeks for the migration of keratinocytes from the basal layer to finally shed off from the SC. The skin also has appendages such as hair follicles and sweat pores, which constitute 0.1% of the total skin surface area. The hair follicles originate from the dermis and terminate at the surface of the skin. Although the SC is only 10 to 20 m thick, it forms a formidable barrier for the exchange of solutes and moisture. It is made up of stacks of keratin-filled corneocytes interdispersed by tightly arranged lipid bilayers (2). The intercellular lipids mainly consist of free fatty acids, ceramides, and cholesterol (2). Molecules can penetrate the skin by three main routes: (i) intracellular (across the corneocytes), (ii) intercellular lipids, and (iii) appendageal [Fig. 1(B)]. The intercellular lipids are the major transport pathways for most drugs, in which the molecule has to pass through successive hydrophilic and hydrophobic domains in the lipid bilayers. On the other hand, the skin appendages serve as a shunt pathway for drug molecules. Since the appendages occupy only a fraction of the skin surface, they contribute very little to the drug transport. However, the appendages constitute a significant pathway for the iontophoretic transport of charged molecules and the penetration of particulate systems (3,4). Once the molecule crosses the SC and subsequently the viable epidermis, it is absorbed through the blood vessels in the dermis [Fig. 1(A)].

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.

Worldwide, breast cancer remains as one of the most common cancer diagnosis and cause of cancer related death among women. Fortunately, nanomedicine has brought forth new potential and hope in breast cancer research. The extremely small size of nanoparticles makes it advantageous and potentially superior to use in tumor detection and imaging. One of the more extensively studied particles is quantum dots, semiconductor crystals which are capable of enhanced labeling and imaging of cancer cells. In addition, due to serious toxicity of chemotherapeutic agents, nano-formulations of breast cancer chemotherapy are under investigation and development. This may provide easier administering route and reduced frequency of drugs. With the use of nanoparticles, drug delivery can be carried out in a minimally invasive fashion and treatment regimens can be made much more targeted and specific for each patient. In this review article, we provide an overview on the role nanomedicine has played in breast cancer and mention some of the latest diagnostic and treatment modalities researched to date.

The main goal of this study is to overcome docetaxel (DTX) resistance in prostate cancer cells by combined delivery of eIF4E siRNA and DTX using dendrimer as a nanocarrier. To this end the objective of this study is to prepare, characterize and test the feasability of the combination delivery approach in-vitro in DTX sensitive and resistant prostate cancer cells. During the last evaluation period we prepared and characterized the dendrimer-DTX conjugate and dendrimer-siRNA complex. During this evaluation period our goal was to optimize the delivery system, develop the DTX resistant prostate cancer cells and test the in-vitro efficacy of the multifunctional delivery system. The DTX resistant prostate cancer cells were developed by treating PC3 cells with sub-therapeutic concentration of DTX for 9 months and it was found to overexpress eIF4E. The multifunctional therapeutic system was able to overcome the resistance in PC3 cells and lowered the IC50 value in drug resistant PC3 cells. The siRNA reduced the levels of eIF4E and Myc in PC3 cells. Overall the outcomes from this study demonstrate the feasibility of developing a therapeutic strategy for drug resistant prostate cancer. Our future studies will focus on further optimizing the delivery system and testing the efficacy in-vivo in prostate cancer mouse model.

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.

Preformulation testing has a significant role in a drug discovery and development program, as it provides valuable feedback to the various discovery and development teams in enabling druggability during lead identification and optimization. In a generic setting, preformulation studies are mainly focused on developing a formulation that is bioequivalent to the innovator's product with the main objective of filing an abbreviated new drug application (ANDA). This article focuses on preformulation testing for oral solid dosage forms in a drug discovery setting. Keywords: preformulation testing; abbreviated new drug application (ANDA); drug discovery; oral solid dosage forms

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