Gemcitabine (Gemzar®) is the first line treatment for pancreatic cancer and often used in combination therapy for non-small cell lung, ovarian, and metastatic breast cancers. Although extremely toxic to a variety of tumor cells in culture, the clinical outcome of gemcitabine treatment still needs improvement. In the present study, a new gemcitabine nanoparticle formulation was developed by incorporating a previously reported stearic acid amide derivative of gemcitabine into nanoparticles prepared from lecithin/glyceryl monostearate-in-water emulsions. The stearoyl gemcitabine nanoparticles were cytotoxic to tumor cells in culture, although it took a longer time for the gemcitabine in the nanoparticles to kill tumor cells than for free gemcitabine. In mice with pre-established model mouse or human tumors, the stearoyl gemcitabine nanoparticles were significantly more effective than free gemcitabine in controlling the tumor growth. PEGylation of the gemcitabine nanoparticles with polyethylene glycol (2000) prolonged the circulation of the nanoparticles in blood and increased the accumulation of the nanoparticles in tumor tissues (> 6-fold), but the PEGylated and un-PEGylated gemcitabine nanoparticles showed similar anti-tumor activity in mice. Nevertheless, the nanoparticle formulation was critical for the stearoyl gemcitabine to show a strong anti-tumor activity. It is concluded that for the gemcitabine derivate-containing nanoparticles, cytotoxicity data in culture may not be used to predict their in vivo anti-tumor activity, and this novel gemcitabine nanoparticle formulation has the potential to improve the clinical outcome of gemcitabine treatment.

An accumulation of research over the years has demonstrated the utility of nanoparticles as antigen carriers with adjuvant activity. Herein we defined the adjuvanticity of a novel lecithin-based nanoparticle engineered from emulsions. The nanoparticles were spheres of around 200 nm. Model protein antigens, bovine serum albumin (BSA) or Bacillus anthracis protective antigen (PA) protein, were covalently conjugated onto the nanoparticles. Mice immunized with the BSA-conjugated nanoparticles developed strong anti-BSA antibody responses comparable to that induced by BSA adjuvanted with incomplete Freund's adjuvant and 6.5-fold stronger than that induced by BSA adsorbed onto aluminum hydroxide. Immunization of mice with the PA-conjugated nanoparticles elicited a quick, strong, and durable anti-PA antibody response that afforded protection of the mice against a lethal dose of anthrax lethal toxin challenge. The potent adjuvanticity of the nanoparticles was likely due to their ability to move the antigens into local draining lymph nodes, to enhance the uptake of the antigens by antigen-presenting cells (APCs), and to activate APCs. This novel nanoparticle system has the potential to serve as a universal protein-based vaccine carrier capable of inducing strong immune responses.

Gemcitabine is a deoxycytidine analog used in the treatment of various solid tumors. However, tumors often develop resistances over time, which becomes a major issue for most gemcitabine-related chemotherapies. In the present study, a previously reported stearoyl gemcitabine nanoparticle formulation (GemC18-NPs) was evaluated for its ability to overcome gemcitabine resistance. In the wild type CCRF-CEM human leukemia cells, the IC50 value of GemC18-NPs was 9.5-fold greater than that of gemcitabine hydrochloride (HCl). However, in the CCRF-CEM-AraC-8C cells that are deficient in the human equilibrative nucleoside transporter-1, the IC50 of GemC18-NPs was only 3.4-fold greater than that in the parent CCRF-CEM cells, whereas the IC50 of gemcitabine HCl was 471-fold greater than that in the parent CCRF-CEM cells. The GemC18-NPs were also more cytotoxic than gemcitabine HCl in the deoxycytidine kinase deficient (CCRF-CEM/dCK−/−) tumor cells. Similar to gemcitabine HCl, GemC18-NPs induced apoptosis through caspase activation. Another gemcitabine-resistant tumor cell line, TC-1-GR, was developed in our laboratory. In the TC-1-GR cells, the IC50 of GemC18-NPs was only 5% of that of gemcitabine HCl. Importantly, GemC18-NPs effectively controlled the growth of gemcitabine resistant TC-1-GR tumors in mice, whereas the molar equivalent dose of gemcitabine HCl did not show any activity against the growth of the TC-1-GR tumors. Proteomics analysis revealed that the TC-1-GR cells over-expressed ribonucleotide reductase M1, which was likely the cause of the acquired gemcitabine resistance in the TC-1-GR cells. To our best knowledge, this presents the first report demonstrating that a nanoparticle formulation of gemcitabine overcomes gemcitabine resistance related to ribonucleotide reductase M1 over-expression.

Docetaxel is a potent anti-cancer drug. However, there continues to be a need for alternative docetaxel delivery systems to improve its efficacy. We reported the engineering of a novel spherical nanoparticle formulation (~270 nm) from lecithin-in-water emulsions. Docetaxel can be incorporated into the nanoparticles, and the resultant docetaxel-nanoparticles were stable when stored as an aqueous suspension. The release of the docetaxel from the nanoparticles was likely caused by a combination of diffusion and Case II transport. The docetaxel-in-nanoparticles were more effective in killing tumor cells in culture than free docetaxel. Moreover, the docetaxel-nanoparticles did not cause any significant red blood cell lysis or platelet aggregation in vitro, nor did they induce detectable acute liver damage when injected intravenously into mice. Finally, compared to free docetaxel, the intravenously injected docetaxel-nanoparticles increased the accumulation of the docetaxel in a model tumor in mice by 4.5-fold. These lecithin-based nanoparticles have the potential to be a novel biocompatible and efficacious delivery system for docetaxel.

There is a growing interest in identifying the relationship between the size of nanoparticles and their adjuvant activity, but the results from recent studies remain controversial. To address the controversy, it was thought that one should pay attention to the nanoparticle formulations to make sure that the antigen-loaded nanoparticles to be compared are not only different in particle size, but more importantly, as identical to each other as possible in all other formulation properties. In the present study, using ovalbumin (OVA) as a model antigen conjugated onto nanoparticles engineered from lecithin/glyceryl monostearate-in-water emulsions, we prepared OVAnanoparticles of 230 nm and 708 nm. Before evaluating the immune responses induced by them in a mouse model, we made sure that: i) the sizes of the two OVA-nanoparticles did not extensively overlap, ii) the nanoparticles have similar zeta potentials and comparable antigen-loading, and iii) the nanoparticles did not aggregate when suspended in simulated biological media. We then showed that when subcutaneously injected into mice, the 230 nm OVA-conjugated nanoparticles induced stronger OVA-specific antibody and cellular immune responses than the 708 nm OVAnanoparticles. Future studies attempting to correlate the size of nanoparticles and their adjuvant activities need to consider formulation parameters to ensure that the particles are different only in size and are stable before and after injection.

Previously, it was shown that a novel 4-(N)-stearoyl gemcitabine nanoparticle formulation was more effective than gemcitabine hydrochloride in controlling the growth of model mouse or human tumors pre-established in mice. In the present study, the feasibility of targeting the stearoyl gemcitabine nanoparticles (GemC18-NPs) into tumor cells that over-express epidermal growth factor receptor (EGFR) to more effectively control tumor growth was evaluated. EGFR is over-expressed in a variety of tumor cells, and EGF is a known natural ligand of EGFR. Recombinant murine EGF was conjugated onto the GemC18-NPs. The ability of the EGF to target the GemC18-NPs to human breast adenocarcinoma cells that expressed different levels of EGFR was evaluated in vitro and in vivo. In culture, the extent to which the EGF-conjugated GemC18-NPs were taken up by tumor cells was correlated to the EGFR density on the tumor cells, whereas the uptake of untargeted GemC18-NPs exhibited no difference among those same cell lines. The relative cytotoxicity of the EGF-conjugated GemC18-NPs to tumor cells in culture was correlated to EGFR expression as well. In vivo, EGFR-over-expressing MDA-MB-468 tumors in mice treated with the EGF-conjugated GemC18-NPs grew significantly slower than in mice treated with untargeted GemC18-NPs, likely due to that the EGF-GemC18-NPs were more anti-proliferative, anti-angiogenic, and pro-apoptotic. Fluorescence intensity data from ex vivo imaging showed that the EGF on the nanoparticles helped increase the accumulation of the GemC18-NPs into MDA-MB-468 tumors pre-established in mice by more than 2-fold as compared to the un-targeted GemC18-NPs. In conclusion, active targeting of the GemC18-NPs into EGFR-over-expressed tumors can further enhance their anti-tumor activity.

This pI:C-adjuvanted rPA vaccine has the potential to be developed into an efficacious nasal anthrax vaccine.

Inhaled anesthetics are believed to produce anesthesia by their actions on ion channels. Because inhaled anesthetics robustly enhance GABA A receptor (GABA A -R) responses to GABA, these receptors are considered prime targets of anesthetic action. However, the importance of GABA A -Rs and individual GABA A -R subunits to specific anesthetic-induced behavioral effects in the intact animal is unknown. We hypothesized that inhaled anesthetics produce amnesia, as assessed by loss of fear conditioning, by acting on the forebrain GABA A -Rs that harbor the ␣1 subunit. To test this, we used global knockout mice that completely lack the ␣1 subunit and forebrain-specific, conditional knockout mice that lack the ␣1 subunit only in the hippocampus, cortex, and amygdala. Both knockout mice were 75 to 145% less sensitive to the amnestic effects of the inhaled anesthetic isoflurane. These results indicate that ␣1-containing GABA A -Rs in the hippocampus, amygdala, and/or cortex influence the amnestic effects of inhaled anesthetics and may be an important molecular target of the drug isoflurane.