William J. Murdoch

Prodrugs Forming High Drug Loading Multifunctional Nanocapsules for Intracellular Cancer Drug Delivery

Anticancer drugs embedded in or conjugated with inert nanocarriers, referred to as nanomedicines, show many therapeutic advantages over free drugs, but the inert carrier materials are the major component (generally more than 90%) in nanomedicines, causing low drug loading contents and thus excessive uses of parenteral excipients. Herein, we demonstrate a new concept directly using drug molecules to fabricate nanocarriers in order to minimize use of inert materials, substantially increase the drug loading content, and suppress premature burst release. Taking advantage of the strong hydrophobicity of the anticancer drug camptothecin (CPT), one or two CPT molecule(s) were conjugated to a very short oligomer chain of ethylene glycol (OEG), forming amphiphilic phospholipid-mimicking prodrugs, OEG-CPT or OEG-DiCPT. The prodrugs formed stable liposome-like nanocapsules with a CPT loading content as high as 40 or 58 wt % with no burst release in aqueous solution. OEG-DiCPT released CPT once inside cells, which showed high in vitro and in vivo antitumor activity. Meanwhile, the resulting nanocapsules can be loaded with a water-soluble drug-doxorubicin salt (DOX.HCl)-with a high loading efficiency. The DOX.HCl-loaded nanocapsules simultaneously delivered two anticancer drugs, leading to a synergetic cytotoxicity to cancer cells. The concept directly using drugs as part of a carrier is applicable to fabricating other highly efficient nanocarriers with a substantially reduced use of inert carrier materials and increased drug loading content without premature burst release.

Maternal Undernutrition from Early- to Mid-Gestation Leads to Growth Retardation, Cardiac Ventricular Hypertrophy, and Increased Liver Weight in the Fetal Sheep

Abstract Early gestation is critical for placentomal growth, differentiation, and vascularization, as well as fetal organogenesis. The fetal origins of adult disease hypothesis proposes that alterations in fetal nutrition and endocrine status result in developmental adaptations that permanently change structure, physiology, and metabolism, thereby predisposing individuals to cardiovascular, metabolic, and endocrine disease in adult life. Multiparous ewes were fed to 50% (nutrient restricted) or 100% (control fed) of total digestible nutrients from Days 28 to 78 of gestation. All ewes were weighed weekly and diets adjusted for individual weight loss or gain. Ewes were killed on Day 78 of gestation and gravid uteri recovered. Fetal body and organ weights were determined, and numbers, morphologies, diameters, and weights of all placentomes were obtained. From Day 28 to Day 78, restricted ewes lost 7.4% of body weight, while control ewes gained 7.5%. Maternal and fetal blood glucose concentrations were reduced in restricted versus control pregnancies. Fetuses were markedly smaller in the restricted group than in the control group. Further, restricted fetuses exhibited greater right- and left-ventricular and liver weights per unit fetal weight than control fetuses. No treatment differences were observed in any gross placentomal measurement. However, caruncular vascularity was enhanced in conceptuses from nutrient-restricted ewes but only in twin pregnancies. While these alterations in fetal/placental development may be beneficial to early fetal survival in the face of a nutrient restriction, their effects later in gestation as well as in postnatal life need further investigation.

Curcumin polymers as anticancer conjugates.

Curcumin has been shown highly cytotoxic towards various cancer cell lines, but its water-insolubility and instability make its bioavailability exceedingly low and thus it generally demonstrates low anticancer activity in in vivo tests. Herein, we report a novel type of polymer-drug conjugates--the high molecular weight curcumin polymers (polycurcumins) made by condensation polymerization of curcumin. The polycurcumins as backbone-type conjugates have advantages of high drug loading efficiency, fixed drug loading contents, stabilized curcumin in their backbones, and tailored water-solubility. The polycurcumins may have many potential applications and their antitumor activities are investigated in this work. The polycurcumins are cytotoxic to cancer cells, but a polyacetal-based polycurcumin (PCurc 8) is highly cytotoxic to SKOV-3, OVCAR-3 ovarian cancers, and MCF-7 breast cancer cell lines. It can be quickly taken up by cancer cells into their lysosomes, where PCurc 8 hydrolyzes and releases active curcumin. It arrests SKOV-3 cell cycle at G(0)/G(1) phase in vitro and induces cell apoptosis partially through the caspase-3 dependent pathway. In vivo, intravenously (i.v.) injected PCurc 8 shows remarkable antitumor activity in SKOV-3 intraperitoneal (i.p.) xenograft tumor model.

Curcumin Inhibits Platelet-Derived Growth Factor–Stimulated Vascular Smooth Muscle Cell Function and Injury-Induced Neointima Formation

Objective—Vascular smooth muscle cell (VSMC) migration, proliferation, and collagen synthesis are key events involved in the pathogenesis of cardiovascular disease. Growth factors, such as platelet-derived growth factor (PDGF) and fibroblast growth factor, released during vascular injury plays a pivotal role in regulating these events. Curcumin (diferuloyl methane), a major component of the spice turmeric (Curcuma longa), has been shown recently to have beneficial effects in chronic conditions, such as inflammation, cancer, cystic fibrosis, and Alzheimer’s disease. The objective of this study was to investigate the ability of curcumin to inhibit PDGF-stimulated migration, proliferation, and collagen synthesis in cultured VSMCs and neointima formation after carotid artery injury in rats. Methods and Results—Curcumin (1 to 25 &mgr;M) produced a concentration-dependent inhibition of PDGF-elicited VSMC migration, proliferation, and collagen synthesis assessed by chemotaxis, [3H]thymidine incorporation, and [3H]-l-proline incorporation, respectively. Curcumin blocked PDGF-induced VSMC actin-cytoskeleton reorganization, attenuated PDGF signal transduction, and inhibited the binding of PDGF to its receptors. Carotid artery neointima formation was significantly attenuated by perivascular curcumin compared with vehicle controls 14 days after injury, characterized by reduced DNA synthesis, collagen synthesis, and PDGF receptor phosphorylation. Conclusions—These data suggest that curcumin is a potent inhibitor of key PDGF-stimulated VSMC functions and may play a critical role in regulating these events after vascular injury.

Oxidative Damage to DNA of Ovarian Surface Epithelial Cells Affected by Ovulation: Carcinogenic Implication and Chemoprevention

The majority of cancers of the ovary are thought to originate from a surface epithelial cell perturbed by ovulation. Outgrowth of a follicle destined to ovulate brings it into apposition with the ovarian epithelium. Ovarian surface cells are consequently exposed, within a limited diffusion radius, to inflammatory agents and reactive oxidants generated during periovulatory processes. Cells that overlie the formative site of follicular rupture suffer irreparable damages and undergo apoptosis. Potentially mutagenic 8-oxoguanine modifications were detected in (surviving) cells circumjacent to postovulatory ovine and human follicles. It is conceivable that clonal expansion of a cell with unrepaired DNA, but not committed to death, could be an initiating factor in the etiology of malignancy, insofar as proliferative ovulatory wound-repair responses may propagate mutations. Since the prognosis for ovarian cancer patients with invasive disease is so poor, and early detection has proven elusive, it is imperative that prospective methods of chemoprevention be explored. Ovulation-induced oxidative base damages to the ovarian epithelium of ewes were prevented by vitamin E. Oxoguanine adducts persisted and CA-125 (a phenotype of metaplastic transformation) was expressed in cultures of cells that were distressed by ovulation in which p53 synthesis was inhibited. Vitamin E negated this reaction. Ovarian cyclicity and fertility were not altered in vitamin-treated ewes. A prophylactic benefit of a supplemental antioxidant is suggested in “ovulating” individuals designated at risk (e.g., due to a tumor suppressor malfunction) for the development of ovarian cancer.

Linear-dendritic drug conjugates forming long-circulating nanorods for cancer-drug delivery

Elongated micelles have many desirable characteristics for cancer-drug delivery, but they are difficult to obtain since amphiphilic polymers form such nanostructures only within narrow composition ranges depending on their own structures. Herein, we demonstrated a facile fabrication of different nanostructures via drug content-controlled self-assembly of amphiphilic linear-dendritic drug conjugates - using the number of the conjugated hydrophobic drug molecule camptothecin (CPT) to tailor the hydrophobicity of amphiphilic PEG-block-dendritic polylysine-CPT (PEG-xCPT) conjugates and thereby control their self-assembled nanostructures - nanospheres or nanorods of different diameters and lengths. The shape and size of the nanostructures were found to strongly affect their in vitro and in vivo properties, particularly the blood clearance kinetics, biodistribution and tumor targeting. The nanorods with medium lengths (<500 nm) had a much longer blood circulation and faster cellular uptake than the nanospheres or long nanorods. Thus, polymeric nanorods with proper lengths may be ideal nanocarriers capable of uniting the opposite requirements in cancer-drug delivery.

Levels of plasma corticosterone and testosterone in male copperheads (Agkistrodon contortrix) following staged fights.

Fighting behavior between male copperheads (Agkistrodon contortrix) occurs during the two mating periods (late summer/fall and spring) to gain priority of access to females. Fights are characterized by prominent vertical challenge displays, swaying, and a high degree of physical contact that does not involve biting. At the moment of subordination, losers retreat quickly from fights and winners respond by chasing. Subsequently, losers do not participate in further challenge displays or fighting for at least 7 days, and also they show behavioral signs of stress, which includes submissive acts and suppression of sexual behavior. The goal of this study was to determine whether or not losers show elevated levels of plasma corticosterone (B) and depressed levels of plasma testosterone (T) relative to winners and controls. Winners and losers were produced in 13 staged trials. Two different controls (N = 26) were run. Males with no recent agonistic experience were (1) tested in the fighting arena in the absence of a competitive male but paired with a single female (N = 13), and (2) tested alone in their cages (N = 13). All trials, including controls, were conducted in spring and late summer. Mean B in losers at 1-hr postfight was significantly greater than in winners and both control groups in both seasons. Mean T was significantly greater in late summer in all groups, as expected, but in each season was not significantly different between the groups. Levels of B and T were not correlated with SVL, mass, or duration of fighting. This study provides further support for the social insensitivity/challenge hypotheses and is the first to document postfight B and T levels in snakes.

Progesterone induces expression of endometrial messenger RNA encoding for cyclooxygenase (sheep).

Exogenous progesterone given early in the ovine estrous cycle results in precocious luteolysis. It has been suggested that under this circumstance regression of the corpus luteum is caused by an advancement in the timing of uterine secretion of prostaglandin F2 alpha. Uterine tissues were obtained from ewes following administration of progesterone (or injection vehicle), fixed in paraformaldehyde, and embedded in paraffin. Tissue sections were hybridized using an 35S-labeled cRNA probe specific for cyclooxygenase mRNA. There was approximately an eight-fold increase in the level of hybridization signal, localized mainly to uterine glands, consequential to treatment with progesterone. Thus, progesterone appears to control expression of the endometrial gene and(or) the stability of the message encoding for a rate-limiting enzyme involved in metabolism of arachidonic acid to its luteolytic product, thereby resolving the length of the nonpregnant cycle.

Amphiphilic curcumin conjugate-forming nanoparticles as anticancer prodrug and drug carriers: in vitro and in vivo effects

Curcumin has been shown to have high cytotoxicity towards various cancer cell lines, but its water insolubility and instability make its bioavailability exceedingly low and, thus, it is generally inactive in in vivo anticancer tests. Here, we report an intracellular-labile amphiphilic surfactant-like curcumin prodrug--curcumin conjugated with two short oligo(ethylene glycol) (Curc-OEG) chains via beta-thioester bonds that are labile in the presence of intracellular glutathione and esterase. Curc-OEG formed stable nanoparticles in aqueous conditions and served two roles--as an anticancer prodrug and a drug carrier. As an anticancer prodrug, the formed nanoparticles had a high and fixed curcumin-loading content of 25.3 wt%, and released active curcumin in the intracellular environment. Curc-OEG had high inhibition ability to several cancer cell lines due to apoptosis. Intravenously injected Curc-OEG significantly reduced the tumor weights and tumor numbers in the athymic mice xenografted with intraperitoneal SKOV-3 tumors and subcutaneous (mammary fat pad) MDA-MB-468 tumors. Preliminary systemic toxicity studies found that Curc-OEG did not cause acute and subchronic toxicities to mouse visceral organs at high doses. As drug carriers, Curc-OEG nanoparticles could carry other anticancer drugs, such as doxorubicin and camptothecin, and ship them into drug-resistant cells, greatly enhancing the cytotoxicity of the loaded drug. Thus, Curc-OEG is a promising prototype that merits further study for cancer therapy.

Charge-reversal polyamidoamine dendrimer for cascade nuclear drug delivery.

AIMS Polyamidoamine (PAMAM) dendrimers with primary amine termini have been extensively explored as drug and gene carriers owing to their unique properties, but their amine-carried cationic charges cause nonspecific cellular uptakes, systemic toxicity and other severe problems in in vivo applications. METHOD In this article, we report a charge-reversal approach that latently deactivates PAMAMs primary amines to negatively charged acid-labile amides in order to inhibit its nonspecific interaction with cells, but regenerates the active PAMAM once in acidic environments. RESULTS A cascade cancer cell nuclear drug delivery was achieved using the latently amidized PAMAM as the carrier conjugated with folic acid as the targeting group and a DNA-toxin drug camptothecin. The conjugate had low nonspecific interactions with cells, but easily entered cancer cells overexpressing folate receptors via receptor-mediated endocytosis. Subsequently, the endocytosed conjugate was transferred to acidic lysosomes, wherein the active PAMAM carrier was regenerated, escaped from the lysosome and then entered the nucleus for drug release. CONCLUSION This reversible deactivation/activation makes PAMAM dendrimers useful nanocarriers for in vivo cancer cell nuclear-targeted drug delivery.