In vitro and in vivo evaluation of Δ9-tetrahidrocannabinol/PLGA nanoparticles for cancer chemotherapy.

Nanoplatforms can optimize the efficacy and safety of chemotherapy, and thus cancer therapy. However, new approaches are encouraged in developing new nanomedicines against malignant cells. In this work, a reproducible methodology is described to prepare Δ(9)-tetrahidrocannabinol (Δ(9)-THC)-loaded poly(d,l-lactide-co-glycolide) (PLGA) nanoparticles against lung cancer. The nanoformulation is further improved by surface functionalization with the biodegradable polymers chitosan and poly(ethylene glycol) (PEG) in order to optimize the biological fate and antitumor effect. Mean nanoparticle size (≈ 290 nm) increased upon coating with PEG, CS, and PEG-CS up to ≈ 590 nm, ≈ 745 nm, and ≈ 790 nm, respectively. Surface electrical charge was controlled by the type of polymeric coating onto the PLGA particles. Drug entrapment efficiencies (≈ 95%) were not affected by any of the polymeric coatings. On the opposite, the characteristic sustained (biphasic) Δ(9)-THC release from the particles can be accelerated or slowed down when using PEG or chitosan, respectively. Blood compatibility studies demonstrated the adequate in vivo safety margin of all of the PLGA-based nanoformulations, while protein adsorption investigations postulated the protective role of PEGylation against opsonization and plasma clearance. Cell viability studies comparing the activity of the nanoformulations against human A-549 and murine LL2 lung adenocarcinoma cells, and human embryo lung fibroblastic MRC-5 cells revealed a statistically significant selective cytotoxic effect toward the lung cancer cell lines. In addition, cytotoxicity assays in A-549 cells demonstrated the more intense anticancer activity of Δ(9)-THC-loaded PEGylated PLGA nanoparticles. These promising results were confirmed by in vivo studies in LL2 lung tumor-bearing immunocompetent C57BL/6 mice.

Fetal cardiac development and hemodynamics in the first trimester.

OBJECTIVE: To describe fetal cardiac and hemodynamic development in the first trimester of pregnancy. SUBJECTS AND METHODS: Forty-eight pregnancies were prospectively studied with transvaginal ultrasound and color Doppler. Fetal heart rate and size were documented together with the presence of inflow and outflow wave-forms and valve signals. RESULTS: Heart rate and size showed a positive correlation with gestational age. At 6 weeks of gestation, 20% of the cardiac cycle was occupied by the isovolumic contraction time (ICT); the isovolumic relaxation time (IRT) occupied 16% and remained unchanged, while the ICT progressively shortened and was not measurable after 12 weeks. The peak inflow velocities had changed from monophasic to biphasic by 10 weeks of gestation. At 7 weeks of gestation, semilunar valves were detected in 10% of examinations and the atrioventricular valves in 3%. The detection rate for both valves at 12 weeks was > or = 90%. In pregnancies that miscarried, the detection rate for both was < 25%. CONCLUSIONS: The Doppler signals that characterize the heart during the first trimester suggest effective heart compliance by 12 weeks. Normal valve development can be inferred from non-invasive Doppler recordings.

Inflammatory changes of the endometrium in patients with minimal-to-moderate endometriosis.

OBJECTIVE: To investigate the endometrium of normal patients for chemotactic activity to neutrophils and macrophages and compare these findings with those of patients with endometriosis. STUDY DESIGN: Endometrial biopsies from patients with and without endometriosis were analyzed for chemotactic activity throughout the menstrual cycle. Glands and stroma of luteal samples were isolated to determine the source of this activity. Infiltrating cells to the endometrium were identified by immunohistochemistry with the use of the monoclonal antibody OKM1. RESULTS: Luteal endometrial samples from normal patients had higher chemotactic activity than samples from the proliferative phase; this was true for both cells types studied: macrophages, 84 versus 10 and neutrophils, 74 versus 11. Patients with endometriosis had high chemotactic activity in both proliferative and luteal biopsies: macrophages, 73 +/- 9 versus 78 +/- 1 and neutrophils, 41 +/- 18 versus 63 +/- 26. Stromal cells from luteal biopsies demonstrated a higher chemotactic activity than the epithelial component. Immunohistochemistry staining identified the infiltrating cells as macrophages. CONCLUSIONS: Extracts from endometrium of normal patients contain chemotactic activity for neutrophils and macrophages; this activity is higher in the secretory phase of the cycle. Endometriosis patients had high chemotactic activity throughout the menstrual cycle. Separation of the endometrial tissue into stroma and the glandular epithelium indicated that the stromal cells are the source of this factor.

Increased chemotactic activity of peritoneal fluid in patients with endometriosis.

OBJECTIVE: Our purpose was to investigate the ability of the peritoneal fluid of patients with endometriosis to induce chemotaxis of neutrophils and macrophages. STUDY DESIGN: Peritoneal fluid samples of patients with endometriosis (n = 20), normal fertile controls (n = 12), or patients with medical suppression (n = 8) were evaluated for chemotactic activity. Results of chemotactic activity were analyzed by analysis of variance. RESULTS: Peritoneal fluid of patients with endometriosis demonstrated a significantly higher chemotactic activity than that of patients without endometriosis or with medical suppression. Patients who had received medical treatment had the lowest chemotactic activity. (p < 0.001 for endometriosis vs control or treatment patients, p = 0.005 for control group vs treatment group). CONCLUSIONS: Patients with endometriosis have a higher chemotactic activity in their peritoneal fluid; prior medical treatment significantly reduces this activity. This chemotactic factor has an estimated weight of 20 kd. The nature and source of this chemotactic factor remains to be determined.

Leukocyte chemotactic activity of FKBP and inhibition by FK506.

Cyclophilin, the cyclosporin A binding protein and member of the immunophilin family of proteins, demonstrates leukocyte chemotactic activity. In this study we demonstrate that FKBP, the FK506 and rapamycin binding protein, also displays leukocyte chemotactic activity. The chemotactic activity of FKBP is inhibited by FK506, however, FK506 was unable to inhibit cyclophilin-stimulated chemotactic activity. Rapamycin was unable to prevent the chemotactic activity of FKBP, similarly, the CsA analogue Me6Ala-CsA while displaying cyclophilin binding was unable to block cyclophilin-stimulated chemotactic activity. These results suggest that in addition to their intracellular role the immunophilins may also function as chemotactic agents, furthermore this activity is modulated by immunosuppressants.

Leukocyte chemotactic activity of cyclophilin.

During the purification of eosinophil chemotactic factors synthesized by the uterus in response to estrogen we isolated a protein having an N-terminal (15 amino acids) sequence identical to that of rat cyclophilin. Our data demonstrate that cyclophilin, a cytosolic protein isolated from bovine thymocytes, which specifically binds the immunosuppressive drug cyclosporin A, as well as recombinant human cyclophilin, displays eosinophil chemotactic activity. In addition to its chemotactic activity, cyclophilin stimulated the release of peroxidase activity from eosinophils. Maximal chemotactic activity of cyclophilin was achieved at a concentration of approximately 10 nM. At similar concentrations cyclophilin was also able to stimulate the migration of neutrophils. This chemotactic activity could be prevented by the addition of cyclosporin A, but not by a nonimmunosuppressive analog (1-fur-furyl-cyclosporin A) at similar concentrations. This chemotactic activity may represent an additional mechanism by which immunosuppressive drugs function to prevent tissue rejection.

Ontogeny of the production of an estrogen-regulated eosinophil chemotactic factor in the rat uterus.

The ontogeny of an estrogen-induced eosinophil chemotactic activity in the uterus after estrogen administration and its regulation by progesterone was studied in neonatal rats at 3, 7, 10, and 13 days of age. These results were compared with the increase in peroxidase activity and the secretion of complement component C3 by the uterus. No chemotactic activity was observed in animals younger than 14 days, and after this age the estrogen regulation of the response was also prevented by the co-administration of progesterone. The secretion of complement C3 appeared earlier, at 10 days of age. It was also observed that the uterus of 3-7-day-old rats responded to estrogen by increasing the secretion of two proteins of 36 and 110 kDa. No further regulation or synthesis was observed with increasing age for these two proteins.

Complement C3 synthesis, peroxidase activity and eosinophil chemotaxis in the rat uterus: effect of estradiol and testosterone.

Treatment of immature rats with estradiol (E2) produced a large increase in uterine peroxidase activity which was accompanied by an increase in eosinophil chemotactic factor (ECF-U). The synthesis of complement C3 was also induced in the uterus and the amount of this 180 kDa protein was determined both by immunoprecipitation and after separation by polyacrylamide gel electrophoresis. Testosterone (T) did not produce an increase in any of these parameters although it antagonized the estrogen-induced increase in uterine peroxidase activity and these effects were more pronounced in estrogen-primed animals. This antagonism was prevented by the antiandrogen, flutamide. Testosterone showed little effect on eosinophil chemotactic activity and did not inhibit the E2-stimulated synthesis of C3. The results with T were supported by the lack of any significant effect by flutamide which antagonizes receptor-mediated androgenic events. These findings are discussed in relation to the action of other types of hormonal steroids (progesterone, dexamethasone) in inhibiting these estrogen-induced molecular changes in the rat uterus and contribute to our understanding of steroid-steroid interaction and the regulation of uterine function.