Combined thermal ablation and liposomal granulocyte-macrophage colony stimulation factor increases immune cell trafficking in a small animal tumor model.

PURPOSE: To characterize intratumoral immune cell trafficking in ablated and synchronous tumors following combined radiofrequency ablation (RFA) and systemic liposomal granulocyte-macrophage colony stimulation factor (lip-GM-CSF). METHODS: Phase I, 72 rats with single subcutaneous R3230 adenocarcinoma were randomized to 6 groups: a) sham; b&c) free or liposomal GM-CSF alone; d) RFA alone; or e&f) combined with blank liposomes or lip-GM-CSF. Animals were sacrificed 3 and 7 days post-RFA. Outcomes included immunohistochemistry of dendritic cells (DCs), M1 and M2 macrophages, T-helper cells (Th1) (CD4+), cytotoxic T- lymphocytes (CTL) (CD8+), T-regulator cells (T-reg) (FoxP3+) and Fas Ligand activated CTLs (Fas-L+) in the periablational rim and untreated index tumor. M1/M2, CD4+/CD8+ and CD8+/FoxP3+ ratios were calculated. Phase II, 40 rats with double tumors were randomized to 4 groups: a) sham, b) RFA, c) RFA-BL and d) RFA-lip-GM-CSF. Synchronous untreated tumors collected at 7d were analyzed similarly. RESULTS: RFA-lip-GMCSF increased periablational M1, CTL and CD8+/FoxP3+ ratio at 3 and 7d, and activated CTLs 7d post-RFA (p<0.05). RFA-lip-GMSCF also increased M2, T-reg, and reduced CD4+/CD8+ 3 and 7d post-RFA respectively (p<0.05). In untreated index tumor, RFA-lip-GMCSF improved DCs, M1, CTLs and activated CTL 7d post-RFA (p<0.05). Furthermore, RFA-lip-GMSCF increased M2 at 3 and 7d, and T-reg 7d post-RFA (p<0.05). In synchronous tumors, RFA-BL and RFA-lip-GM-CSF improved DC, Th1 and CTL infiltration 7d post-RFA. CONCLUSION: Systemic liposomal GM-CSF combined with RFA improves intratumoral immune cell trafficking, specifically populations initiating (DC, M1) and executing (CTL, FasL+) anti-tumor immunity. Moreover, liposomes influence synchronous untreated metastases increasing Th1, CTL and DCs infiltration.

Biocompatible ionic liquids assisted transdermal co-delivery of antigenic protein and adjuvant for cancer immunotherapy.

Human skin contains numerous antigen-presenting cells that are a potential target for several immune-based therapies, including vaccination and cancer immunotherapy. However, the outermost layer of the skin-the stratum corneum-acts as a major physical barrier against the permeation of antigens that have a molecular weight > 500 Da. In this study, an ionic liquid-assisted delivery system (ILDS) was developed, which enabled the successful transdermal delivery of an antigenic protein, ovalbumin (OVA), with a toll-like receptor agonist, imiquimod, as an adjuvant, to stimulate a specific immune response. Both the ionic liquids and ILDS were completely biocompatible for topical or transdermal application for therapeutic purposes. The skin permeation of the antigenic protein and adjuvant was found to be significantly enhanced because of the incorporation of a surface-active ionic liquid in the ILDS. An in vivo immunization study showed that there was a high level of OVA-specific IgG antibody production because of the enhanced permeation of the antigen and adjuvant across and into the skin. In a preclusive anticancer study, vaccination through ILDS showed stronger tumor-growth inhibition compared to control group. These results indicated that the ILDS could be a promising strategy for transdermal immunization as future therapeutics.

Lipid-Based Ionic-Liquid-Mediated Nanodispersions as Biocompatible Carriers for the Enhanced Transdermal Delivery of a Peptide Drug.

Lipid-based biocompatible ionic liquids (LBILs) have attracted attention as carriers in transdermal drug delivery systems (TDDSs) because of their lipophilic character. In this study, we report the formulation of a peptide-LBIL complex microencapsulated in an oil phase as a potential carrier for the transdermal delivery of leuprolide acetate as a model hydrophilic peptide. The peptide-LBIL complexes were prepared via a water-in-oil emulsion composed of 1,2-dimyristoyl-sn-glycerol-3-ethyl-phosphatidylcholine (EDMPC), a fatty acid (stearic, oleic, and linoleic acid)-based LBIL, and cyclohexane followed by freeze-drying to remove the water and cyclohexane. Then, the peptide-LBIL complexes were nanodispersed and stabilized in isopropyl myristate (IPM) using sorbitol laurate (Span-20). Ionic-liquid-in-oil nanodispersions (IL/O-NDs) were prepared with varying weight ratios of LBILs and Span-20 as the surfactant and the cosurfactant, respectively. Keeping the overall surfactant constant at 10 wt % in IPM, a 5:5 wt % ratio of surfactant (IL) and cosurfactant (Span-20) in the IL/O-NDs significantly (p < 0.0001) increased the physiochemical stability, drug-loading capacity, and drug encapsulation efficiency. The in vitro and in vivo peptide delivery across the skin was increased significantly (p < 0.0001) using IL/O-NDs, compared with non-IL-treated groups. Of all of the LBIL-based formulations, [EDMPC][Linoleate]/O-ND was considered the most preferable for a TDDS based on the pharmacokinetic parameters. The transdermal delivery flux with [EDMPC][Linoleate]/O-ND was increased 65-fold compared with the aqueous delivery vehicle. The IL/O-NDs were able to deform the lipid and protein arrangements of the skin layers to enhance the transdermal permeation of the peptide. In vitro and in vivo cytotoxicity studies of the IL/O-NDs revealed the biocompatibility of the LBIL-based formulations. These results indicated that IL/O-NDs are promising biocompatible carriers for lipid-peptide TDDSs.

Ionic Liquid-In-Oil Microemulsions Prepared with Biocompatible Choline Carboxylic Acids for Improving the Transdermal Delivery of a Sparingly Soluble Drug.

The transdermal delivery of sparingly soluble drugs is challenging due to of the need for a drug carrier. In the past few decades, ionic liquid (IL)-in-oil microemulsions (IL/O MEs) have been developed as potential carriers. By focusing on biocompatibility, we report on an IL/O ME that is designed to enhance the solubility and transdermal delivery of the sparingly soluble drug, acyclovir. The prepared MEs were composed of a hydrophilic IL (choline formate, choline lactate, or choline propionate) as the non-aqueous polar phase and a surface-active IL (choline oleate) as the surfactant in combination with sorbitan laurate in a continuous oil phase. The selected ILs were all biologically active ions. Optimized pseudo ternary phase diagrams indicated the MEs formed thermodynamically stable, spherically shaped, and nano-sized (<100 nm) droplets. An in vitro drug permeation study, using pig skin, showed the significantly enhanced permeation of acyclovir using the ME. A Fourier transform infrared spectroscopy study showed a reduction of the skin barrier function with the ME. Finally, a skin irritation study showed a high cell survival rate (>90%) with the ME compared with Dulbecco's phosphate-buffered saline, indicates the biocompatibility of the ME. Therefore, we conclude that IL/O ME may be a promising nano-carrier for the transdermal delivery of sparingly soluble drugs.

In vivo biocompatibility, pharmacokinetics, antitumor efficacy, and hypersensitivity evaluation of ionic liquid-mediated paclitaxel formulations.

In order to prevent common hypersensitivity reactions to paclitaxel injections (Taxol), we previously reported an ionic liquid-mediated paclitaxel (IL-PTX) formulation with small particle size and narrow size distribution. The preliminary work showed high PTX solubility in the IL, and the formulation demonstrated similar antitumor activity to Taxol, while inducing a smaller hypersensitivity effect in in vitro cell experiments. In this study, the stability of the IL-PTX formulation was monitored by quantitative HPLC analysis, which showed that IL-PTX was more stable at 4 °C than at room temperature. The in vivo study showed that the IL-PTX formulation could be used in a therapeutic application as a biocompatible component of a drug delivery system. To assess the in-vivo biocompatibility, IL or IL-mediated formulations were administered intravenously by maintaining physiological buffered conditions (neutral pH and isotonic salt concentration). From in vivo pharmacokinetics data, the IL-PTX formulation was found to have a similar systemic circulation time and slower elimination rate compared to cremophor EL mediated paclitaxel (CrEL-PTX). Furthermore, in vivo antitumor and hypersensitivity experiments in C57BL/6 mice revealed that IL-PTX had similar antitumor activity to CrEL-PTX, but a significantly smaller hypersensitivity effect compared with CrEL-PTX. Therefore, the IL-mediated formulation has potential to be an effective and safe drug delivery system for PTX.

Ionic-Liquid-Based Paclitaxel Preparation: A New Potential Formulation for Cancer Treatment.

Paclitaxel (PTX) injection (i.e., Taxol) has been used as an effective chemotherapeutic treatment for various cancers. However, the current Taxol formulation contains Cremophor EL, which causes hypersensitivity reactions during intravenous administration and precipitation by aqueous dilution. This communication reports the preliminary results on the ionic liquid (IL)-based PTX formulations developed to address the aforementioned issues. The formulations were composed of PTX/cholinium amino acid ILs/ethanol/Tween-80/water. A significant enhancement in the solubility of PTX was observed with considerable correlation with the density and viscosity of the ILs, and with the side chain of the amino acids used as anions in the ILs. Moreover, the formulations were stable for up to 3 months. The driving force for the stability of the formulation was hypothesized to be the involvement of different types of interactions between the IL and PTX. In vitro cytotoxicity and antitumor activity of the IL-based formulations were evaluated on HeLa cells. The IL vehicles without PTX were found to be less cytotoxic than Taxol, while both the IL-based PTX formulation and Taxol exhibited similar antitumor activity. Finally, in vitro hypersensitivity reactions were evaluated on THP-1 cells and found to be significantly lower with the IL-based formulation than Taxol. This study demonstrated that specially designed ILs could provide a potentially safer alternative to Cremophor EL as an effective PTX formulation for cancer treatment giving fewer hypersensitivity reactions.

Comparison of direct versus concentrated smear microscopy in detection of pulmonary tuberculosis.

BACKGROUND: Sputum smear microscopy is fast and inexpensive technique for detecting tuberculosis (TB) in high incidence areas but has low sensitivity. Physical and chemical sputum processing along with centrifugation have been found to show promise in overcoming this limitation. Our objective was to compare the sensitivity of smear microscopy obtained with smears made directly from respiratory specimens to those from concentrated specimens. METHODS: By active screening, 915 TB suspects were identified from Dhaka Central Jail and sputum specimens were aseptically collected. Direct smears were prepared by taking a small portion of the purulent part of the sputum with a sterile loop. The specimens were then processed by a standard N-acetyl-L-cysteine-NaOH digestion-decontamination method to prepare concentrated specimens. Both smears were then air dried, heat fixed, and stained by the Ziehl-Neelsen staining technique. The stained slides were examined under oil immersion and were graded following International Union Against Tuberculosis and Lung Diseases guidelines. All the specimens were inoculated into Lowenstein-Jensen (L-J) media and culture results were considered as gold standard to calculate sensitivity. RESULTS: Of 915 specimens, 73 (8%) specimens were positive both on direct and concentrated methods, one sample was positive on direct microscopy but was negative on concentrated method. An extra 14 (1.5%) samples were positive on concentrated method which were negative on direct smear. In L-J media 105 specimens were found positive for TB bacilli and of them, 74 (70.5%) and 87 (82.9%) were positive in direct and concentrated smear, respectively. The sensitivity of direct and concentrated smear microscopy was different when using positive culture as the gold standard (71% vs. 83%). CONCLUSIONS: The results showed that concentrated technique increases the sensitivity of microscopy up to 12%. Therefore, the national programs in high TB burden countries may consider incorporating the technique into their guidelines at least in the district and higher level laboratories to improve case finding strategy.