Molecularly precise dendrimer-drug conjugates with tunable drug release for cancer therapy.

The structural preciseness of dendrimers makes them perfect drug delivery carriers, particularly in the form of dendrimer-drug conjugates. Current dendrimer-drug conjugates are synthesized by anchoring drug and functional moieties onto the dendrimer peripheral surface. However, functional groups exhibiting the same reactivity make it impossible to precisely control the number and the position of the functional groups and drug molecules anchored to the dendrimer surface. This structural heterogeneity causes variable pharmacokinetics, preventing such conjugates to be translational. Furthermore, the highly hydrophobic drug molecules anchored on the dendrimer periphery can interact with blood components and alter the pharmacokinetic behavior. To address these problems, we herein report molecularly precise dendrimer-drug conjugates with drug moieties buried inside the dendrimers. Surprisingly, the drug release rates of these conjugates were tailorable by the dendrimer generation, surface chemistry, and acidity.

Acid-active cell-penetrating peptides for in vivo tumor-targeted drug delivery.

Cell-penetrating peptides (CPPs) such as transactivator of transcription (TAT) peptide have long been explored for promoting in vitro cell penetration and nuclear targeting of various cargos, but their positive charges cause strong nonspecific interactions, making them inapplicable for many in vivo applications. In this work, we used TAT to demonstrate a molecular modification approach for inhibiting nonspecific interactions of CPPs in the bloodstream while reactivating their functions in the targeted tissues or cells. The TAT lysine residues' amines were amidized to succinyl amides ((a)TAT), completely inhibiting TAT's nonspecific interactions in the blood compartment; once in the acidic tumor interstitium or internalized into cell endo/lysosomes, the succinyl amides in the (a)TAT were quickly hydrolyzed, fully restoring TAT's functions. Thus, (a)TAT-functionalized poly(ethylene glycol)-block-poly(ε-caprolactone) micelles achieved long circulation in the blood compartment and efficiently accumulated and delivered doxorubicin to tumor tissues, giving rise to high antitumor activity and low cardiotoxicity. This amidization strategy effectively and easily enables in vivo applications of CPPs.

Maternal nutrient restriction in the ewe from early to midgestation programs reduced steroidogenic enzyme expression and tended to reduce progesterone content of corpora lutea, as well as circulating progesterone in nonpregnant aged female offspring.

BACKGROUND: Previously we reported decreased circulating progesterone and fertility in one and two year old ewes born to undernourished mothers. This study was designed to investigate if this reduction in progesterone persisted into old age, and if it did, what mechanisms are involved. METHODS: Ewes were fed a nutrient restricted (NR, 50% of NRC recommendations) or control (C, 100% of NRC) diets from day 28 to 78 of gestation, then all were fed to requirements through parturition and weaning. Female offspring (4 per treatment group) were maintained as a group and fed to requirements from weaning until assigned to this study at 6 years of age. Ewes were synchronized for estrus (day 0) and blood samples were collected daily from day 0 to day 11 before necropsy on day 12. Blood serum and luteal tissue were assayed for progesterone concentrations by validated radioimmunoassay. RESULTS: Circulation progesterone concentrations tended to be lower (P = 0.06) in NR than C offspring from day 0 to 11 of the estrous cycle. While total luteal weight was similar across groups, total progesterone content also tended to be reduced (P = 0.07) in luteal tissue of NR than C offspring. Activity of hepatic progesterone catabolizing enzymes and selected angiogenic factors in luteal tissue were similar between groups. Messenger RNA expression of steroidogenic enzymes StAR and P450scc were reduced (P < 0.05), while protein expression of StAR tended to be reduced (P < 0.07) and P450scc was reduced (P < 0.05) in luteal tissue of NR versus C offspring. CONCLUSIONS: There appears to be no difference in hepatic steroid catabolism that could have led to the decreased serum progesterone. However, these data are consistent with the programming of decreased steroidogenic enzyme expression in CL of NR offspring, leading to reduced synthesis and secretion of progesterone.

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.

"I fired my last doctor for not taking me seriously": collaborating with a difficult medical patient in a multidisciplinary primary care facility.

We present the case of a multidisciplinary primary care assessment of a 32-year-old woman with multiple medical and psychological complaints. Following the collaborative care model, this assessment was conducted by a team consisting of a clinical health psychologist, Dr. J. L. Skillings, and a family physician, Dr. W. J. Murdoch. We describe the primary care environment in which this referral was made including the methods that were utilized to insure a successful professional collaboration. We report the results and recommendations from a comprehensive biopsychosocial assessment; we place emphasis on the psychological diagnosis and pain symptoms. We also describe the feedback session in which the assessment results were provided to the patient and her spouse by both physician and psychologist. Multiperspective commentary about the assessment is offered by the patient and her husband as well as the physician and psychologist assessors.

Degradable poly(beta-amino ester) nanoparticles for cancer cytoplasmic drug delivery.

Fast cytoplasmic drug delivery can overcome cancer cells' drug resistance and thus have an enhanced therapeutic efficacy. Such a drug delivery regime requires drug carriers capable of entering cancer cells, localizing and rapidly releasing the drug into endosomes/lysosomes, and subsequently disrupting their membranes to release the drug into the cytosol. We herein report a low-toxic and degradable poly(beta-amino ester)-graft-polyethylene glycol (BAE-PEG) co-polymer forming pH-responsive nanoparticles capable of cytoplasmic drug delivery. BAE-PEG was synthesized by condensation polymerization of diacrylate and piperazine in the presence of a PEG-diacrylate macromonomer. BAE-PEG with 2% or 5% PEG side chains formed micelles (nanoparticles) with diameters of about 100 nm. The BAE-PEG nanoparticles were shown to rapidly enter cancer cells, localize in their endosomes/lysosomes, and subsequently disrupt them to release the drugs into the cytosol. Camptothecin loaded in the nanoparticles had a higher cytotoxicity to SKOV-3 ovarian cancer cells than free camptothecin.

Progesterone facilitates cisplatin toxicity in epithelial ovarian cancer cells and xenografts.

OBJECTIVES: The underlying premise of these investigations was that the lipophilic hormone progesterone, which partitions into and (at relatively high concentrations) impedes the fluid mechanics of the plasmalemma, would perturb integral associations between membrane lipids and exporter pumps that otherwise confer drug resistance. That progesterone can affect susceptibility of ovarian adenocarcinoma cells and xenografts to cisplatin was tested. METHODS: The cisplatin-resistant human cell lines SKOV-3 and OVCAR-3 were treated for 24 hours with cisplatin (0.1 microg/ml)+/-progesterone (0.01, 0.1 microg/ml). Cytotoxicity and platinum were measured by MTT assay and inductively coupled plasma mass spectrometry, respectively. Athymic mice were inoculated intraperitoneal (ip) with SKOV-3 cells. Cisplatin (2 mg/kg/week)+/-progesterone (25 mg sustained-release pellet) regimens were initiated ip at one week (when micrometastases were present) and continued to six weeks post-xenograft. Tumor burdens, histopathology, and platinum concentrations were assessed upon necropsy at 24 hours after the final injection of cisplatin. RESULTS: There were no significant in vitro/vivo anticancer effects of cisplatin alone. High-dose progesterone enhanced platinum accretion and induced drug toxicity in both cell lines. Tumorigenesis was suppressed by cisplatin+progesterone. The treatment synergy was related to elevated tumor platinum and morphological evidence of apoptosis. CONCLUSION: It appears that the addition of progesterone to ovarian cancer therapeutic modalities represents a step in improving responses.

pH-responsive nanoparticles for cancer drug delivery.

Solid tumors have an acidic extracellular environment and an altered pH gradient across their cell compartments. Nanoparticles responsive to the pH gradients are promising for cancer drug delivery. Such pH-responsive nanoparticles consist of a corona and a core, one or both of which respond to the external pH to change their soluble/insoluble or charge states. Nanoparticles whose coronas become positively charged or become soluble to make their targeting groups available for binding at the tumor extracellular pH have been developed for promoting cellular targeting and internalization. Nanoparticles whose cores become soluble or change their structures to release the carried drugs at the tumor extracellular pH or lysosomal pH have been developed for fast drug release into the extracellular fluid or cytosol. Such pH-responsive nanoparticles have therapeutic advantages over the conventional pH-insensitive counterparts.

Morphologic responses of the mouse ovarian surface epithelium to ovulation and steroid hormonal milieu.

Ovarian cancer of surface epithelial origin is an ovulation- and endocrine-related disease. It appears that a cell transformed by genotoxins generated at follicular rupture is propagated during postovulatory wound repair. A consequent steroid hormonal imbalance favoring the mitogenic estrogens is a prospective predisposing factor in ovarian neoplasia. Protection against epithelial ovarian cancer is conferred by progesterone. The objective of this study was to characterize the acute effects of ovulation and steroid hormonal exposure on morphologic responses of surface epithelial cells of mouse ovaries. Follicular development and ovulation were induced in immature animals with equine and human (=Day 0) choriogonadotropins, respectively. On Day 2 (approximately 36 hrs after ovulation), surface epithelial classifications presented in histologic sections were altered from simple (single-layered) squamous and cuboidal toward stratification; this trend was reversed (i.e., reverted to the control status) on Days 4-8. Shifts in the ovarian epithelium from simple to stratified were accentuated following postovulatory (Days 1-8) treatment with estradiol. Surface epithelia of ovaries obtained after 1 week of progesterone administration were exclusively of a simple phenotype. We conclude that the proliferative/procarcinogenic reaction of the ovarian surface epithelium to ovulation is exacerbated by estrogen and counteracted by progesterone.

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 microM) 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.

Synthesis of degradable functional poly(ethylene glycol) analogs as versatile drug delivery carriers.

Poly(ethylene glycol) (PEG) is widely used as a water soluble carrier for polymer-drug conjugates. Herein, we report degradable linear PEG analogs (DPEGs) carrying multifunctional groups. The DPEGs were synthesized by a Michael addition based condensation polymerization of dithiols and PEG diacrylates (PEGDA) or dimethacrylates (PEGDMA). They were stable at pH 7.4 but quickly degraded at pH 6.0 and 5.0. Thus, DPEGs could be used as drug carriers without concern for their retention in the body. DPEGs could be made to carry such functional groups as terminal thiol or (meth)acrylate and pendant hydroxyl groups. The functional groups were used for conjugation of drugs and targeting groups. This new type of PEG analog will be useful for drug delivery and the PEGylation of biomolecules and colloidal particles.

Degradable cisplatin-releasing core-shell nanogels from zwitterionic poly(beta -aminoester)-graft-PEG for cancer chemotherapy.

Cisplatin conjugated onto macromolecules or loaded in micelles can be preferentially delivered to tumors to minimize its toxicity to healthy tissues and increase its drug efficacy. Herein, we report cisplatin-containing nanogels possibly useful for targeted delivery of cisplatin. Carboxylic acid-functionalized poly(beta -aminoester)graft-poly(ethylene glycol) copolymers were synthesized by cocondensation polymerization of piperazine with 2,2-bis(acryloxymethyl)propionic acid, PEG 2,2-bis(acryloxymethyl)propionate macromonomer (mPEG). The graft copolymers formed 100-200 nm nanogels with low size-distribution by the complexation of their carboxylic groups with cisplatin. The nanogels were negatively charged and had a PEG outer layer. Thus, they had "stealth properties" suitable for in vivo applications. The nanogels had significantly lower in vitro cytotoxicity to SKOV-3 ovarian cancer cells than free cisplatin, but similar anticancer activity toward SKOV-3 tumors xenografted to immunocompromised mice.

Complement-inhibiting effect of ovarian cancer antigen CA-125.

Malignant transformation of ovarian cells of surface epithelial origin is associated with expression of a membrane-spanning glycoprotein, cancer antigen (CA)-125. The bulk of the putative CA-125 molecule is comprised a very large, folded, multivalent, mucin-like exodomain. That the extracellular motif of CA-125 exerts immunosuppressive effects which promote tumor progression has been suggested. We report that CA-125 attenuates complement lysis of antibody-sensitized cells. The secreted form of CA-125 derived from culture medium of the human ovarian adenocarcinoma cell line OVCAR-3 caused a dose-response inhibition of sheep erythrocyte hemolysis. Moreover, OVCAR-3 cells became prone to complement attack (trypan blue uptake) mediated by a gonadotropin-releasing hormone receptor antibody when (membrane-bound) CA-125 was excised/removed by trypsin/washing; this effect was counteracted by replacement with (soluble) CA-125. It is conceivable that CA-125 entraps/sheds effectors of the complement cascade.

Highly stable core-surface-crosslinked nanoparticles as cisplatin carriers for cancer chemotherapy.

Cisplatin is a potent anticancer drug with low solubility in water. A new type of highly stable polymer micelles, namely core-surface-crosslinked nanoparticles (SCNPs) made from amphiphilic brush copolymers, were evaluated as the carrier of cisplatin. Cisplatin could be loaded in the SCNPs with poly(epsilon-caprolactone) (PCL) cores and hydrophilic poly(ethylene glycol) (PEG) or poly[2-(N,N-dimethylamino)ethyl methacrylate] (PDMA) shells with high loading efficiency (approximately 90%). In vitro cellular uptake experiments indicated that both SCNPs could be easily taken up by SKOV-3 ovarian cancer cells. Both cell proliferation assay and IC50 measurements indicated that cisplatin encapsulated in the SCNPs had much enhanced cytotoxicity to the cancer cells compared to free cisplatin. The positive charges on the PCL/PDMA SCNPs promoted the cellular internalization of the nanoparticles, resulting in higher cytotoxicity of cisplatin in these SCNPs. The IC50 of the cisplatin encapsulated in PCL/PDMA SCNPs was as low as 0.01 microg/mL, lower than that of cisplatin in PCL/PEG SCNPs and free cisplatin.

Fabrication of dendrimer-releasing lipidic nanoassembly for cancer drug delivery.

An inherent dilemma in the use of nanomedicines for cancer drug delivery is their limited penetration into tumors due to their large size. We have demonstrated that dendrimer/lipid nanoassemblies can solve this problem by means of tumor-triggered disassembly and the release of small (several nanometers) dendrimers to facilitate tumor penetration. Herein, we report a general strategy for the fabrication of nanoassemblies from hydrophobic and hydrophilic dendrimers with phospholipids. Hydrophobic dendrimers could assemble with lipids via hydrophobic interactions, whereas hydrophilic dendrimers could only assemble with lipids in the presence of anionic surfactants via both electrostatic and hydrophobic interactions. The nanoassemblies of hydrophobic dendrimers/lipids were found to be capable of stripping off their lipid layers via fusion with the cell membrane and then intracellular or extracellular release of dendrimers, whereas the nanoassemblies of hydrophilic dendrimers/lipids were internalized via endocytosis and then released their dendrimers inside the cells. Therefore, these dendrimer/lipid nanoassemblies could be used for the delivery of different cancer drugs.

Effects of progesterone on ovarian tumorigenesis in xenografted mice.

Circumstantial evidence indicates that progestins reduce the risk of epithelial ovarian cancer. We report that the tumorigenic capacity of human ovarian carcinoma (SKOV-3) cells inoculated into the peritoneal cavity of athymic mice is suppressed by pretreatment with subcutaneous progesterone-releasing pellets. Numbers of tumor implants on the intestines/mesentery and invasiveness into underlying host tissues were reduced at 6 weeks following exposure to progesterone. Progesterone prevented tumors from forming on the liver. Life spans of progesterone-treated animals were prolonged. There was no beneficial effect of administration of progesterone if initiated after ovarian tumors had become established on organ surfaces. Our findings implicate a role for progesterone in ovarian cancer prophylaxis.

DNA damages in ovarian surface epithelial cells of ovulatory hens.

A cause-effect relationship between ovulation and common (surface) epithelial ovarian cancer has been suspected for many years. The ovarian surface epithelium apparently becomes exposed to genotoxins that are generated during the ovulatory process. Intensive egg-laying hens readily develop ovarian carcinomatosis. Indeed, elevated levels of potentially mutagenic 8-oxo-guanine adducts were detected in avian ovarian epithelial cells isolated from the apical surfaces and perimeters of pre-and postovulatory follicles, respectively. Internucleosomal DNA fragmentation indicative of apoptosis was evident in ovarian surface epithelial cells associated with the formative site of ovulation (stigma line) and regressive ruptured follicles. It is conceivable that a genetically altered progenitor cell with unrepaired DNA but not committed to death (i.e., a unifocal "escape") could give rise to a transformed phenotype. Hence, the high rate of ovarian cancer in egg-laying hens could be the consequence of genomic damages to the ovarian surface epithelium associated with incessant ovulations, thereby increasing the likelihood of mutation and clonal expansion.

A multifunctional PEG-PLL drug conjugate forming redox-responsive nanoparticles for intracellular drug delivery.

Tumor-targeting, redox-responsive and high drug-loaded nanoparticles were synthesized from poly(ethylene glycol)-b-poly(l-lysine) (PEG-PLL) for enhanced cancer therapy. A hydrophobic drug camptothecin (CPT) was anchored to the lysine residual amines in PEG-PLL via disulfide bonds. Folate acid as targeting group was further introduced to the PLL block via long PEG chains. The conjugate self-assembled into nanoparticles of around 100 nm with hydrophobic CPT moieties forming the core and folate acid targeting groups on the shell. The nanoparticles were expected to be stable in the blood circulation but once internalized via folate receptor-mediated endocytosis, disintegrate and release the drug by glutathione in the cytosol. The nanoparticles could be used as a nanocarrier to further encapsulate other drugs such as doxorubicin for combined chemotherapy. The CPT-conjugated nanoparticles had comparable cytotoxicity to free CPT at low doses but higher cytotoxicity than CPT at high doses.

Metaplastic potential of p53 down-regulation in ovarian surface epithelial cells affected by ovulation.

That ovulation is a predisposing factor in common (surface) epithelial ovarian cancer is widely recognized; however, the molecular events which underscore early-stage disease have not been elucidated. In vivo and in vitro studies using an ovine model system were designed to address a premise that oxidative distresses to DNA inflicted upon ovarian surface epithelial cells within a limited diffusion radius of the ovulatory site of follicular rupture, if gone uncorrected by p53-dependent cycle arrest/repair pathways, could yield a progenitor of tumorigenic potential. Immunofluorescence image analysis was used to quantitate the DNA damage marker 8-oxoguanine, the tumor suppressor p53, the base-excision repair polymerase beta, and apoptotic internucleosomal DNA fragmentation in ovarian surface epithelial cells isolated from the perimeter of ovulated follicles. Up-regulation of p53 coincided with accretion of 8-oxoguanine lesions. Oxidative disturbances to DNA were reconciled during the consequent luteal phase (before replicative repair of the ovarian rupture wound). Production of p53 was not related to apoptosis, but rather to induction of polymerase beta. Oxoguanine modifications persisted in cells affected by ovulation in which synthesis of p53 was negated in culture by an antisense oligonucleotide. Inhibition of p53 was associated with discordant cellular growth rates and expression of the cancer antigen CA-125 - a phenotype of metaplastic transformation. It is suggested that the integrity of DNA of ovarian surface epithelial cells is compromised by reactive oxidants and inflammatory mediators generated during the ovulatory process and that malfunction in a damage-recognition and(or) repair mechanism is a determinant in the etiology of ovarian metaplasia and carcinogenesis.

Steroid hormonal regulation of proliferative, p53 tumor suppressor, and apoptotic responses of sheep ovarian surface epithelial cells.

Ovarian surface epithelial cells have been implicated in the genesis of common ovarian cancers. The integrity of DNA of ovarian surface epithelial cells contiguous with the ovulatory stigma becomes compromised during the rupture process; most cells degenerate by apoptosis, however some, bearing sublethal lesions, persist along the margins of ovulated follicles. Clonal expansion of a genetically-damaged surface epithelial cell (i.e. with unrepaired DNA, but not committed to death) can presumably give rise to ovarian carcinoma. It was hypothesized that estradiol and progesterone regulate ovarian surface epithelial cell-cycle dynamics associated with folliculo-luteal transitions and ovulatory wound repair/remodeling. Progesterone up-regulated the tumor suppressor p53 and inhibited baseline and estradiol-stimulated proliferation of cultured sheep ovarian surface epithelial cells. Anti/mitotic responses to steroid hormones were transcriptionally- and receptor-dependent. Rates of apoptosis (DNA fragmentation) were unaffected by progesterone. High concentrations of estradiol, via a nongenomic (perhaps antioxidant) mechanism, suppressed basal and H(2)O(2)-induced apoptosis. We suggest that, progesterone serves to inhibit proliferation of ovarian surface epithelial cells throughout the luteal phase--providing the time (growth arrest) required to correct any metabolic disturbances to DNA that are perpetrated as an inevitable by-product of the ovulatory process. With luteolysis and dominance of an estrogenic preovulatory follicle the ovarian surface epithelium is then regenerated. Thus, it is conceivable that perturbations to the steroid hormonal milieu of ovarian cycles could be a predisposing factor for cancerous transformation of an ovarian surface epithelial cell.