The genomic revolution has identified therapeutic targets for a plethora of diseases, creating a need to develop robust technologies for combination drug therapy. In the present work, we describe a self-assembled polymeric nanoparticle (NP) platform to target and control precisely the codelivery of drugs with varying physicochemical properties to cancer cells. As proof of concept, we codelivered cisplatin and docetaxel (Dtxl) to prostate cancer cells with synergistic cytotoxicity. A polylactide (PLA) derivative with pendant hydroxyl groups was prepared and conjugated to a platinum(IV) [Pt(IV)] prodrug, c,t,c-½PtðNH 3 Þ 2 ðO 2 CCH 2 CH 2 COOHÞðOHÞCl 2 [PLAPt(IV)]. A blend of PLA-Pt(IV) functionalized polymer and carboxylterminated poly(D,L-lactic-co-glycolic acid)-block-poly(ethylene glycol) copolymer in the presence or absence of Dtxl, was converted, in microfluidic channels, to NPs with a diameter of ∼100 nm. This process resulted in excellent encapsulation efficiency (EE) and high loading of both hydrophilic platinum prodrug and hydrophobic Dtxl with reproducible EEs and loadings. The surface of the NPs was derivatized with the A10 aptamer, which binds to the prostatespecific membrane antigen (PSMA) on prostate cancer cells. These NPs undergo controlled release of both drugs over a period of 48-72 h. Targeted NPs were internalized by the PSMA-expressing LNCaP cells via endocytosis, and formation of cisplatin 1,2-d(GpG) intrastrand cross-links on nuclear DNA was verified. In vitro toxicities demonstrated superiority of the targeted dual-drug combination NPs over NPs with single drug or nontargeted NPs. This work reveals the potential of a single, programmable nanoparticle to blend and deliver a combination of drugs for cancer treatment.chemotherapy | drug delivery | polymer-drug conjugate | targeting | temporal release

Steroidogenic factor-1 (SF-1) (Ad4BP, NR5A1) is a nuclear receptor that regulates many aspects of adrenal and reproductive development and function. Consequently, deletion of the gene (Nr5a1) encoding Sf-1 in XY mice results in impaired adrenal development, complete testicular dysgenesis with Müllerian structures, and female external genitalia. Initial efforts to identify NR5A1 changes in humans focused on 46,XY individuals with combined adrenogonadal failure and Müllerian structures. Although this combination of clinical features is rare, 2 such patients harboring NR5A1 mutations have been described within the past decade. More recently, however, it has emerged that heterozygous loss of function mutations in NR5A1 can be found relatively frequently in children and adults with 46,XY disorders of sex development (DSD) but with apparently normal adrenal function. The phenotypic spectrum associated with these changes ranges from complete testicular dysgenesis with Müllerian structures, through individuals with mild clitoromegaly or genital ambiguity, to severe penoscrotal hypospadias or even anorchia. Furthermore, a nonsynonymous polymorphism in NR5A1 may be associated with micropenis or undescended testes within the population. Taken together, these reports suggest that variable loss of SF-1 function can be associated with a wide range of reproductive phenotypes in humans. KeywordsGonad; Gonadal dysgenesis; NR5A1; Nuclear receptor; Pituitary; Steroidogenesis; Steroidogenic Factor-1; 46,XY DSD The Identification and Cloning of NR5A1The concept of a common 'steroidogenic factor' that could activate multiple different steps in steroidogenesis was first proposed in the early 1990s following the identification of a number of similar regulatory elements in the proximal promoter regions of the cytochrome P450 steroid hydroxylase genes [Rice et al., 1991;Morohashi et al., 1992]. These elements contained variations on an AGGTCA DNA sequence motif, leading to the hypothesis that a common protein -termed 'steroidogenic factor-1' (SF-1) -could regulate their transcription.The gene encoding Sf-1 in the mouse (now termed Nr5a1) was first cloned in 1992 from an adrenal cDNA library using a probe corresponding to the DNA-binding domain (DBD) of a related orphan receptor, retinoid X receptor [Lala et al., 1992]. The bovine homologue of this factor (termed adrenal 4-binding protein, Ad4BP) was identified shortly afterwards from an adrenal cDNA library using the partial sequence of a protein purified from bovine adrenal extracts [Honda et al., 1993]. Both these murine and bovine cDNAs were shown to encode proteins that could activate the promoters of steroid hydroxylase enzymes. Thus, it was concluded that a common steroidogenic factor had been identified.The mouse gene encoding steroidogenic factor-1 was initially termed FtzF1, as it resembles the Drosophila orphan nuclear receptor, fushi tarazu factor homolog 1 (FTZ-F1) [Ueda et al., 1990;Swift and Ashworth, 1995;Taketo et al., 1995]. This gene was mapped to chromosome 2. ...

It has long been assumed that the red cell membrane is highly permeable to gases because the molecules of gases are small, uncharged, and soluble in lipids, such as those of a bilayer. as expected, but also appeared to reduce intracellular A, although separate experiments showed it has no effect on CA activity in homogenous solution. A decrease in P m,CO 2 would explain this finding. With a more advanced computational model, which solves for CA activity and membrane permeabilities to both CO 2 and HCO 3 ؊ , we found that DIDS inhibited both P m,HCO ؊ 3 and P m,CO 2 , whereas intracellular CA activity remained unchanged. The mechanism by which DIDS reduces CO 2 permeability may not be through an action on the lipid bilayer itself, but rather on a membrane transport protein, implying that this is a normal route for at least part of red cell CO 2 exchange. Ϫ and H 2 16 O in a red cell suspension at chemical equilibrium can be used in principle to measure the proportional intracellular acceleration (A) of CO 2 hydration produced inside intact red blood cells by carbonic anhydrase (CA) compared with the uncatalyzed rate (1) and the self-exchange permeability of the membrane to HCO 3 (P m,HCO Ϫ 3 ). As predicted, A was found to be the same in intact erythrocytes as in hemolysate, both under normal conditions and when exposed to varying concentrations of a membrane-permeable CA sulfonamide inhibitor (ethoxzolamide), whereas P m,HCO Ϫ 3 was not affected (2). Validation of the ability of the technique to differentiate between CA activity and HCO 3 Ϫ permeability in intact red cells was extended by exposing them to phlorizin (3), an established Band III inhibitor (1); this drug decreased P m,HCORecently we exposed red cells to a more specific inhibitor of Band III protein, 4,4Ј-diisothiocyanato-stilbene-2,2Ј-disulfonate (DIDS), to decrease P m,HCO Ϫ 3 without inhibiting CA. However, we found that whereas DIDS lowered HCO 3 Ϫ transport, it also lowered the membrane permeability to CO 2 . METHODSTwo groups of experiments were carried out, one in Philadelphia and one in Hannover, Germany. Blood from healthy adults, freshly drawn into heparin, was washed three times with 145 mM NaCl at 4°C; the red cells were diluted to approximately 40% hematocrit and used the same day. Hemolysates were prepared by freezing and thawing the cell suspension.The fractional water volume, v, of the red cells in the reaction suspension was calculated as the hematocrit of the stock cell suspension ϫ water content of red cells [0.61 (1) for the Philadelphia experiments and 0.65 (4) for the Hannover experiments] ϫ its dilution in the reaction mixture. In later experiments, v was calculated as 55.5 ϫ [cyanmethemoglobin] in mM in the reactant mixture. NaHCO 3 was prepared by incubating 2% and 5% 18O labeled HOH with unlabeled NaHCO 3 at 150°C in a pressure bomb for several days, after which the water was removed by lyophylization. DIDS was obtained from Research Organics and phlorizin (phloridzin) was obtained from Sigma.The technique of the ...