Hydroxychloroquine (HCQ) has been shown to demonstrate anti-inflammatory properties and direct anti-HIV activity. In this study, we describe for the first time the fabrication and in vitro evaluation of two types of intravaginal ring (IVR) devices (a surfaced-modified matrix IVR and a reservoir segmental IVR) for achieving sustained delivery (>14 days) of HCQ as a strategy for preventing male-to-female transmission of HIV. Both IVRs were fabricated by hot-melt injection molding. Surface-modified matrix IVRs with polyvinylpyrrolidone or poly(vinyl alcohol) coatings exhibited significantly reduced burst release on the first day (6.45% and 15.72% reduction, respectively). Reservoir IVR segments designed to release lower amounts of HCQ displayed near-zero-order release kinetics with an average release rate of 28.38 µg/mL per day for IVRs loaded with aqueous HCQ and 32.23 µg/mL per day for IVRs loaded with HCQ mixed with a rate-controlling excipient. Stability studies demonstrated that HCQ was stable in coated or noncoated IVRs for 30 days. The IVR segments had no significant effect on cell viability, pro-inflammatory cytokine production, or colony formation of vaginal and ectocervical epithelial cells. Both IVR systems may be suitable for the prevention of HIV transmission and other sexually transmitted infections.
The goal of this study was to develop and characterize an intravaginal nanomedicine for the active targeted delivery of saquinavir (SQV) to CD4+ immune cells as a potential strategy to prevent or reduce HIV infection. The nanomedicine was formulated into a vaginal gel to provide ease in self-administration and to enhance retention within the vaginal tract. SQV-encapsulated nanoparticles (SQV-NPs) were prepared from poly(lactic-co-glycolic acid)(PLGA) and conjugated to antihuman anti-CD4 antibody. Antibody-conjugated SQV-NPs (Ab-SQV-NPs) had an encapsulation efficiency (EE%) of 74.4% ± 3.7% and an antibody conjugation efficiency (ACE%) of 80.95% ± 1.10%. Over 50% of total loaded SQV was released from NPs over 3 days. NPs were rapidly taken up by Sup-T1 cells, with more than a twofold increase in the intracellular levels of SQV when delivered by Ab-SQV-NPs in comparison to controls 1 hour post-treatment. No cytotoxicity was observed when vaginal epithelial cells were treated for 24 hours with drug-free Ab-NPs (1,000 µg/mL), 1% HEC placebo gel (200 mg/mL), or 1% HEC gel loaded with drug-free Ab-NPs (5 mg NPs/g gel, 200 mg/mL of gel mixture). Overall, we described an intravaginal nanomedicine that is nontoxic and can specifically deliver SQV into CD4+ immune cells. This platform may demonstrate potential utility in its application as postexposure prophylaxis for the treatment or reduction of HIV infection, but further studies are required.
The vaginal tract is a suitable site for the administration of both local and systemic acting drugs. There are numerous vaginal products on the market such as those approved for contraception, treatment of yeast infection, hormonal replacement therapy, and feminine hygiene. Despite the potential in drug delivery, the vagina is a complex and dynamic organ that requires greater understanding. The recent discovery that injections of double stranded RNA (dsRNA) in Caenorhabditis elegans (C. elegans) results in potent gene specific silencing, was a major scientific revolution. This phenomenon known as RNA interference (RNAi), is believed to protect host genome against invasion by mobile genetic elements such as transposons and viruses. Gene silencing or RNAi has opened new potential opportunities to study the function of a gene in an organism. Furthermore, its therapeutic potential is being investigated in the field of sexually transmitted infections such as human immunodeficiency virus (HIV) and other diseases such as age-related macular degeneration (AMD), diabetes, hypercholesterolemia, respiratory disease, and cancer. This review will focus on the therapeutic potential of siRNA for the treatment and/or prevention of infectious diseases such as HIV, HPV, and HSV within the vaginal tract. Specifically, formulation design parameters to improve siRNA stability and therapeutic efficacy in the vaginal tract will be discussed along with challenges, advancements, and future directions of the field.
Polyethylene glycol (PEG) has been used widely in liposomal formulations as a strategy to inhibit opsonization by plasma proteins and to prolong liposome plasma circulation time. PEG can be incorporated onto the surface of liposomes either during the spontaneous self-assembling process or inserted after vesicle formation. The advantages of employing the PEG postinsertion method include improved drug encapsulation efficiency and the ability to incorporate PEG conjugates for enhanced cell binding and uptake. In this study, we propose to evaluate a cationic lipid nanoparticle formulation containing two PEGylation steps: pre- and post-siRNA insertion. Our results indicate that formulations consisting of the extra PEG post-insertion step significantly increased siRNA circulation in the plasma by two-folds in comparison with the formulations consisting of only the single PEGylation step. Moreover, this formulation was able to efficiently carry siRNA to the tumor site, increase siRNA stability and significantly downregulate luciferase mRNA expression by >50% when compared with the controls in an intraperitoneal and subcutaneous breast cancer tumor model. Overall, our cationic lipid nanoparticle formulation displayed enhanced plasma circulation, reduced liver accumulation, enhanced tumor targeting, and effective gene knockdown--demonstrating excellent utility for the delivery of siRNA.
Cationic liposomes exhibit a propensity to selectively target tumor-associated blood vessels demonstrating potential value as anti-cancer drug delivery vehicles. Their utility however, is hampered by their biological instability and rapid elimination following i.v. administration. Efforts to circumvent rapid plasma elimination have, to date, focused on decreasing cationic lipid content and incorporating polyethylene glycol (PEG)-modified lipids. In this study we wanted to determine whether highly charged cationic liposomes with surface-associated PEG could be designed to exhibit extended circulation lifetimes, while retaining tumor vascular targeting properties in an HT29 colorectal cancer xenograft model. Cationic liposomes prepared of DSPC, cationic lipids (DODAC, DOTAP, or DC-CHOL), and DSPE-PEG(2000) were studied. Our results demonstrate that formulations prepared with 50 mol% DODAC or DC-CHOL, and 20 mol% DSPE-PEG(2000) exhibited circulation half-lives ranging from 6.5 to 12.5 h. Biodistribution studies demonstrated that DC-CHOL formulations prepared with DSPE-PEG(2000) accumulated threefold higher in s.c. HT29 tumors than its PEG-free counterpart. Fluorescence microscopy studies suggested that the presence of DSPE-PEG(2000) did not adversely affect liposomal tumor vasculature targeting. We show for the first time that it is achievable to design highly charged, highly pegylated (20 mol% DSPE-PEG(2000)) cationic liposomes which exhibit both extended circulation lifetimes and tumor vascular targeting properties.
Localized and sustained delivery of anti-cancer agents to the tumor site has great potential for the treatment of solid tumors. A chitosan–egg phosphatidylcholine (chitosan–ePC) implant system containing PLA-b-PEG/PLA nanoparticles has been developed for the delivery of paclitaxel to treat ovarian cancer. Production of volumes of ascites fluid in the peritoneal cavity is a physical manifestation of ovarian cancer. In vitro release studies of paclitaxel from the implant were conducted in various fluids including human ascites fluid. A strong correlation (r2 = 0.977) was found between the release of paclitaxel in ascites fluid and PBS containing lysozyme (pH 7.4) at 37 °C. The drug release mechanism for this system was proposed based on swelling, degradation and morphology data. In addition, in vitro release of paclitaxel was found to be a good indicator of the in vivo release profile (correlation between release rates: r2 = 0.965). Release of paclitaxel was found to be sustained over a four-week period following implantation of the chitosan–ePC system into the peritoneal cavity of healthy Balb/C mice. Also, the concentrations of paclitaxel in both plasma and tissues (e.g. liver, kidney and small intestine) were found to be relatively constant.
Endotoxin-induced inflammation alters the hepatic expression of the drug efflux transporter genes mdr1b (Abcb1b) and mrp3 (Abcc3) in rats. In this study, we identified a novel kruppel-like zinc finger protein 6 (KLF6) cis-element on the rat mdr1b promoter which is important for basal activity and IL-1beta and endotoxin-mediated induction in gene transcription. Interestingly, KLF6 also functioned as a negative transcriptional regulator, inhibiting TNF-alpha-mediated induction of mdr1b. Furthermore, novel CCAAT/enhancer binding protein beta (C/EBPbeta) and heat shock factor 4 (HSF4) transcription binding sites were identified on the rat mrp3 promoter. Deletion of the HSF4 element significantly increased transcriptional activity of the mrp3 gene when exposed to TNF-alpha. Endotoxin treatment significantly affected transcriptional activity only in C/EBPbeta and HSF4 double deletion mrp3 promoter constructs. In summary, KLF6 and HSF4 are stimuli-specific regulatory elements which may be important in the control of the rat mdr1b and mrp3 genes during health and disease.
Recently, we developed a novel implantable drug delivery system which can provide sustained intraperitoneal (i.p.) delivery of paclitaxel (PTX). As the impact of local sustained delivery on the development of multidrug resistance (MDR) is unknown, the objective of this study was to determine the impact of this drug delivery system on the in vivo expression of MDR1/P-glycoprotein (PGP) in a human ovarian xenograft tumor model. As compared to controls, intermittent i.p. dosing with PTX formulated in Cremophor EL (PTX(CrEL)) induced a two-fold increase in mRNA levels of MDR1 after a 14-day dosing period. On the other hand, sustained i.p. delivery of PTX with the implant system (PTX(film)) did not significantly affect MDR1 expression. Immunodetection of PGP in isolated xenografts supported the mRNA data. Histological analysis by H&E staining demonstrated a dose-dependent increase in tumor necrosis in the PTX(film) treated animals. Further, in vitro studies in human ovarian carcinoma cells also demonstrated a significant induction in the efflux activity of PGP with intermittent dosing schedules to PTX(CrEL) whereas this was not seen in cells dosed with PTX(film). Our findings suggest that sustained i.p. administration with PTX(film) attenuates development of MDR, suggesting that sustained, localized delivery of chemotherapeutic agents may improve current treatment strategies for ovarian cancer.
DNA frayed wires (DNA(FW)) are an alternate form of DNA organization formed by the self-association of several strands of guanine-rich oligonucleotides. The purpose of this study was to define for the first time the blood clearance kinetics, tissue distribution, and stability of DNA(FW) in vivo in mice. Single bolus doses (1200 pmol/mouse) of (32)P-DNA(FW) and (32)P-random DNA were administered intravenously (IV) and intraperitoneally (IP) followed by scheduled blood, urine, fecal and tissue samplings. Blood clearance kinetics was described well by a first order two-compartment open model. The overall half-lives of elimination from the central compartment (T(1/2))(K10) were 3.57+/-0.1h for IV and 2.38+/-0.11 h for IP. In contrast, random DNA was completely degraded after 15 min regardless of the route of administration. Tissue distribution results demonstrated that DNA(FW) were primarily distributed and retained in the liver, intestines, kidneys, and heart. Low levels could also be detected in brain. Autoradiographs of blood, tissues, feces and urine extracts established that DNA(FW) remained intact after administration as no measurable levels of metabolites or degradation products were found after 24h. (32)P-DNA(FW) was primarily eliminated via hepato-biliary excretion into feces after either IV or IP administration (51.8+/-4.53% and 36.2+/-3.4%, respectively). The improved stability and longer half-life of DNA(FW), previously shown in vitro, is also seen in vivo, indicating that DNA(FW) may provide a stable delivery system for DNA gene therapies. In conclusion, this is the first study demonstrating the in vivo stability, pharmacokinetics, and disposition of DNA superstructures.
Various mechanisms have been implicated in the development of resistance of cancer cells to chemotherapy. Multidrug resistance (MDR) is a phenomenon in which cancer cells are resistant to the cytotoxic effects of various structurally and mechanistically unrelated chemotherapeutic agents. One major mechanism by which this occurs is through the over-expression of ATP-dependent drug efflux transporters such as the P-glycoprotein (PGP) and multidrug resistance-associated protein (MRP). Regulation of MDR can occur at many levels including transcriptional, mRNA, protein and post-translational. In recent years it has been demonstrated that alterations in the expression and activity of the MDR transporters are seen in numerous tissues during an inflammatory response. An acute inflammatory response is associated with many conditions including infection, injury, hypoxia and stress and is known to result in the induction of several pro-inflammatory cytokines. Whether the function of cytokines can be harnessed in overcoming drug resistance of tumors has yet to be examined and explored. In this review, we will focus on the various studies investigating the regulation of MDR during an inflammatory response, in particular by cytokines. The mediators and pathways involved as well as the possible mechanisms of MDR regulation will be discussed. It is hoped that by understanding the clinical importance of inflammatory mediators in MDR, new doors will open and future insights will lead to the development of novel immunotherapeutics for the treatment of cancer.
Recently, a novel chitosan-based implantable formulation (chitosan-ePC) was developed to provide controlled, local release of paclitaxel (PTX) for the treatment of ovarian tumors. Hence, the objective of this study was to evaluate this delivery system in vitro in human ovarian SKOV-3 cells and in vivo in mice with intraperitoneal implants of drug-free or 14C-PTX-chitosan-ePC films. In vitro, 14C-PTX-chitosan-ePC implants (10 mg) provided zero-order constant release of 0.92+/-0.03 pg/day PTX over 5 days. Released PTX retained dose-dependent activity; effectively inhibiting SKOV-3 proliferation with an ED50 of 211 ng/ml of released PTX. Drug-free implants did not affect cell viability or cell morphology of SKOV-3 cells. A sustained, zero-order release of PTX was also seen in vivo over a 2 week period in mice implanted with 14C-PTX-chitosan-ePC films. Correlations between the in vitro and in vivo release of PTX was highly significant (R2 = 0.975). After 2-4 weeks, mice with chitosan-ePC implants did not demonstrate any signs of encapsulation, inflammation or infection. Overall, our in vitro and in vivo results demonstrated zero-order drug release and biocompatibility of the novel chitosan-ePC film. This indicates potential usefulness of chitosan-ePC implants in the sustained and local delivery of anti-neoplastic agents.
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