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Biomedical subjects

James R Baker

Publications and source records attributed to James R Baker.

At least 19 recordsLinked to original sources
PubMed2024

Enhanced mucosal SARS-CoV-2 immunity after heterologous intramuscular mRNA prime/intranasal protein boost vaccination with a combination adjuvant.

Current COVID-19 mRNA vaccines delivered intramuscularly (IM) induce effective systemic immunity, but with suboptimal immunity at mucosal sites, limiting their ability to impart sterilizing immunity. There is strong interest in rerouting immune responses induced in the periphery by parenteral vaccination to the portal entry site of respiratory viruses, such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), by mucosal vaccination. We previously demonstrated the combination adjuvant, NE/IVT, consisting of a nanoemulsion (NE) and an RNA-based RIG-I agonist (IVT) induces potent systemic and mucosal immune responses in protein-based SARS-CoV-2 vaccines administered intranasally (IN). Herein, we demonstrate priming IM with mRNA followed by heterologous IN boosting with NE/IVT adjuvanted recombinant antigen induces strong mucosal and systemic antibody responses and enhances antigen-specific T cell responses in mucosa-draining lymph nodes compared to IM/IM and IN/IN prime/boost regimens. While all regimens induced cross-neutralizing antibodies against divergent variants and sterilizing immunity in the lungs of challenged mice, mucosal vaccination, either as homologous prime/boost or heterologous IN boost after IM mRNA prime, was required to impart sterilizing immunity in the upper respiratory tract. Our data demonstrate the benefit of hybrid regimens whereby strong immune responses primed via IM vaccination are rerouted by IN vaccination to mucosal sites to provide optimal protection against SARS-CoV-2.

Animals↗
PubMed2007

Dendrimer-based targeted delivery of an apoptotic sensor in cancer cells.

Our previous studies have demonstrated the applicability of poly(amidoamine) (PAMAM) dendrimers as a platform for the targeted delivery of chemotherapeutic drugs both in vitro and in vivo. To monitor the rate and extent of cell-killing caused by the delivered chemotherapeutic drug, we wished to analyze the degree of apoptosis in targeted cells on a real-time basis. As the apoptosis-regulating caspases are activated during the apoptotic process, several caspase-hydrolyzable, fluorescence resonance energy transfer (FRET)-based substrates have been marketed for the detection of apoptosis. However, the applicability of these agents is limited because of their nonspecificity and the consequent high background fluorescence in tissues. Here we show the synthesis, characterization, and in vitro targeting of an engineered PAMAM nanodevice in which folic acid (FA) is conjugated as the targeting molecule and a caspase-specific FRET-based agent (PhiPhiLux G1D2) is conjugated as the apoptosis-detecting agent. This conjugate specifically targets FA-receptor-positive, KB cells. In these cells, the apoptosis-inducing agent staurosporine caused a 5-fold increase in the cellular fluorescence. These results show, for the first time, the potential applicability of a targeted apoptosis-measuring nanodevice, which could be used for simultaneously monitoring the apoptotic potential of a delivered drug.

Apoptosis↗
PubMed2006

HPLC analysis of functionalized poly(amidoamine) dendrimers and the interaction between a folate-dendrimer conjugate and folate binding protein.

Poly(amidoamine) (PAMAM) dendrimers of different generations with carboxyl, acetyl, and hydroxyl terminal groups and a folic acid (FA)-dendrimer conjugate were separated and analyzed using reverse-phase high performance liquid chromatography (HPLC). Analysis of both the individual PAMAM derivatives and the separation of mixed generations can be achieved using a linear gradient 0-50% acetonitrile (ACN) (balance water) within 40 min. We also show that PAMAMs with defined acetylation and carboxylation degrees can be analyzed using HPLC. Furthermore, a generation 5 dendrimer-FA conjugate (G5.75Ac-FA4; Ac denotes acetyl) was analyzed and its specific binding with a bovine folic acid binding protein (FBP) was monitored. The HPLC and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) results indicate the formation of three complexes after the binding of G5.75Ac-FA4 with FBP. Dendrimers with FA moieties show much higher specific binding capability with FBP than those without FA moieties. Findings from this study indicate that HPLC is an effective technique not only for characterization and separation of functionalized PAMAM dendrimers and conjugates but also for investigation of the interaction between dendrimers and biomolecules.

Biocompatible Materials↗
PubMed2006

Molecular dynamics studies of the size, shape, and internal structure of 0% and 90% acetylated fifth-generation polyamidoamine dendrimers in water and methanol.

We have performed molecular dynamics simulations of 0% and 90% acetylated fifth-generation (G5) polyamidoamine (PAMAM) dendrimers in water and methanol and obtained radii of gyration of 2.51-2.57 and 2.11-2.33 nm, respectively, similar to those measured experimentally in methanol by Prosa et al. (J. Polym. Sci. 1997, 35, 2913-2924) and in water by Choi et al. (Nano Lett. 2004, 4, 391-397). Calculation of the moments of inertia and the relative shape anisotropy show that both 0% and 90% acetylated G5 are modestly ellipsoidal. The distribution of branch points relative to the center of the dendrimer and penetration of solvent show that the core and surface of the dendrimer are more exposed to water than is the region between the core and the surface due to the interactions between monomers, including hydrogen bonds that rapidly break and re-form as solvent molecules compete for hydrogen-bonding sites on the monomers. The water and methanol solvents seem to produce similar numbers of hydrogen bond interactions between monomers.

Acetylation↗
PubMed2006

Molecular heterogeneity analysis of poly(amidoamine) dendrimer-based mono- and multifunctional nanodevices by capillary electrophoresis.

Poly(amidoamine) (PAMAM) dendrimer-based nanodevices are of recent interest in targeted cancer therapy. Characterization of mono- and multifunctional PAMAM-based nanodevices remains a great challenge because of their molecular complexity. In this work, various mono- and multifunctional nanodevices based on PAMAM G5 (generation 5) dendrimer were characterized by UV-Vis spectrometry, (1)H NMR, size exclusion chromatography (SEC), and capillary electrophoresis (CE). CE was extensively utilized to measure the molecular heterogeneity of these PAMAM-based nanodevices. G5-FA (FA denotes folic acid) conjugates (synthesized from amine-terminated G5.NH(2) dendrimer, approach 1) with acetamide and amine termini exhibit bimodal or multi-modal distributions. In contrast, G5-FA and bifunctional G5-FA-MTX (MTX denotes methotrexate) conjugates with hydroxyl termini display a single modal distribution. Multifunctional G5.Ac(n)-FI-FA, G5.Ac(n)-FA-OH-MTX, and G5.Ac(n)-FI-FA-OH-MTX (Ac denotes acetamide; FI denotes fluorescein) nanodevices (synthesized from partially acetylated G5 dendrimer, approach 2) exhibit a monodisperse distribution. It indicates that the molecular distribution of PAMAM conjugates largely depends on the homogeneity of starting materials, the synthetic approaches, and the final functionalization steps. Hydroxylation functionalization of dendrimers masks the dispersity of the final PAMAM nanodevices in both synthetic approaches. The applied CE analysis of mono- and multifunctional PAMAM-based nanodevices provides a powerful tool to evaluate the molecular heterogeneity of complex dendrimer conjugate nanodevices for targeted cancer therapeutics.

Amines↗
PubMed2006

Electrophoretic mobility and molecular distribution studies of poly(amidoamine) dendrimers of defined charges.

Generation 5 ethylenediamine (EDA)-cored poly(amidoamine) (PAMAM) dendrimers (E5, E denotes the EDA core and 5 the generation number) with different degrees of acetylation and carboxylation were synthesized and used as a model system to investigate the effect of charge and the influence of dendrimer surface modifications on electrophoretic mobility (EM) and molecular distribution. The surface-modified dendrimers were characterized by size-exclusion chromatography, 1H NMR, MALDI-TOF-MS, PAGE, and CE. The focus of our study was to determine how EM changes as a function of particle charge and molecular mass, and how the molecular distribution changes due to surface modifications. We demonstrate that partially modified dendrimers have much broader migration peaks than those of fully surface functionalized or unmodified E5 dendrimers due to variations in the substitution of individual dendrimer surfaces. EM decreased nonlinearly with increases in surface acetylation for both PAMAM acetamides and PAMAM succinamic acids, indicating a complex migration activity in CE separations that is not solely due to charge/mass ratio changes. These studies provide new insights into dendrimer properties under an electric field, as well as into the characterization of dendrimer-based materials being developed for medical applications.

Acetylation↗
PubMed2006

PAMAM dendrimer-based multifunctional conjugate for cancer therapy: synthesis, characterization, and functionality.

Poly(amidoamine) (PAMAM) dendrimer-based multifunctional cancer therapeutic conjugates have been designed and synthesized. The primary amino groups on the surface of the generation 5 (G5) PAMAM dendrimer were neutralized through partial acetylation, providing enhanced solubility of the dendrimer (in conjugation of FITC (fluorescein isothiocyanate)) and preventing nonspecific targeting interactions (in vitro and in vivo) during delivery. The functional molecules fluorescein isothiocyanate (FITC, an imaging agent), folic acid (FA, targets overexpressed folate receptors on specific cancer cells), and paclitaxel (taxol, a chemotherapeutic drug) were conjugated to the remaining nonacetylated primary amino groups. The appropriate control dendrimer conjugates have been synthesized as well. Characterization of the G5 PAMAM dendrimer and its nanosize conjugates, including the molecular weight and number of primary amine groups, has been determined by multiple analytical methods such as gel permeation chromatography (GPC), nuclear magnetic resonance spectroscopy (NMR), potentiometric titration, high-performance liquid chromatography (HPLC), and UV spectroscopy. These multifunctional dendrimer conjugates have been tested in vitro for targeted delivery of chemotherapeutic and imaging agents to specific cancer cells. We present here the synthesis, characterization, and functionality of these dendrimer conjugates.

Antineoplastic Agents↗
PubMed2006

No apparent damage in the thyroid of transgenic mice expressing antiapoptotic FLIP.

FLIP is an antiapoptotic protein that has been demonstrated to play an important role in inflammation, cancer, and autoimmune diseases. However, it is not known whether increased expression of FLIP (FLICE inhibitory protein) in thyrocytes would alter the development of the thyroid and/or pathogenesis of thyroiditis. To examine the effects of overexpression of this antiapoptotic molecule on the thyroid, we have developed transgenic mouse lines that specifically express FLIP in thyrocytes. A DNA construct designed with an in-frame coding sequence for the E8 protein, a viral FLIP, was put under the control of the thyroglobulin (Tg) promoter (the Tg-FLIP transgene). In 8 of 12 resultant transgenic mouse lines, FLIP expression in thyrocytes driven by the Tg promoter was documented, and confirmed at RNA and protein levels. These Tg-FLIP transgenic mice were monitored for 1 year. Throughout the entire observation period, the transgenic mice remained alive and healthy without evidence of thyroid dysfunction. Adult mice were able to breed. Histologic examination of thyroids obtained at various time points did not reveal significant differences between transgenic mice and their control littermates. Therefore, transgenic mice with thyrocyte-specific expression of FLIP have normal thyroid development with no significant changes in thyroid cell death or proliferation.

Animals↗
PubMed2006

Diffusion of Alexa Fluor 488-conjugated dendrimers in rat aortic tissue.

In this study, the distribution of labeled dendrimers in native and aneurysmal rat aortic tissue was examined. Adult male rats underwent infrarenal aorta perfusion with generation 5 (G5) acetylated Alexa Fluor 488-conjugated dendrimers for varying lengths of time. In a second set of experiments, rats underwent aortic elastase perfusion followed by aortic dendrimer perfusion 7 days later. Aortic diameters were measured prior to and postelastase perfusion, and again on the day of harvest. Aortas were harvested 0, 12, or 24 h postperfusion, fixed, and mounted. Native aortas were harvested and viewed as negative controls. Aortic cross-sections were viewed and imaged using confocal microscopy. Dendrimers were quantified (counts/high-powered field). Results were evaluated by repeated measures ANOVA and Student's t-test. We found that in native aortas, dendrimers penetrated the aortic wall in all groups. For all perfusion times, fewer dendrimers were present as time between dendrimer perfusion and aortic harvest increased. Longer perfusion times resulted in increased diffusion of dendrimers throughout the aortic wall. By 24 h, the majority of the dendrimers were through the wall. Dendrimers in aneurysmal aortas, on day 0 postdendrimer perfusion, diffused farther into the aortic wall than controls. In conclusion, this study documents labeled dendrimers delivered intra-arterially to native rat aortas in vivo, and the temporal diffusion of these molecules within the aortic wall. Increasing perfusion time and length of time prior to harvest resulted in continued dendrimer diffusion into the aortic wall. These preliminary data provide a novel mechanism whereby local inhibitory therapy may be delivered locally to aortic tissue.

Aneurysm↗
PubMed2006

Efficacy assessment of Aphidius colemani (Hymenoptera: Braconidae) for suppression of Aphis gossypii (Homoptera: Aphididae) in greenhouse-grown chrysanthemum.

To assess biological control as a management tool for the cotton aphid, Aphis gossypii Glover (Homoptera: Aphididae), the efficacy of Aphidius colemani Viereck (Hymenoptera: Braconidae) for suppression of A. gossypii in greenhouse-grown chrysanthemums, Dendranthema grandiflora (Tzvelev), was compared with a pesticide standard, imidacloprid (Marathon 1% G) and an untreated check. No significant differences were found between aphid populations in the two treatments. A. colemani and imidacloprid kept aphid numbers very low, with the correspondent aphid populations exhibiting very low intrinsic rates of increase (r(m) = -0.0369 and r(m) = 0.0151, respectively), in contrast to the exponential growth of aphid populations (r(m) = 0.1085) observed on the untreated plants. Parasitism levels in A. colemani plots ranged from 48.93 to 83.38%. Esthetic damage parameters, including exuviae, honeydew, and sooty mold on leaves, were significantly different between treatments and untreated control, and damage levels were minimal with the insecticide treatment and natural enemy releases. The cost of A. colemani releases was 4.7 times greater than the cost of the imidacloprid treatment.

Animals↗
PubMed2005

Targeted drug delivery with dendrimers: comparison of the release kinetics of covalently conjugated drug and non-covalent drug inclusion complex.

Dendrimers have unique characteristics including monodispersity and modifiable surface functionality, along with highly defined size and structure. This makes these polymers attractive candidates as carriers in drug delivery applications. Drug delivery can be achieved by coupling a drug to polymer through one of two approaches. Hydrophobic drugs can be complexed within the hydrophobic dendrimer interior to make them water-soluble or drugs can be covalently coupled onto the surface of the dendrimer. Using both methods we compared the efficacy of generation 5 PAMAM dendrimers in the targeted drug delivery of methotrexate coupled to the polymer. The amine-terminated dendrimers bind to negatively charged membranes of cells in a non-specific manner and can cause toxicity in vitro and in vivo. To reduce toxicity and to increase aqueous solubility, modifications were made to the surface hydroxyl groups of the dendrimers. For targeted drug delivery, the dendrimer was modified to have a neutral terminal functionality for use with surface-conjugated folic acid as the targeting agent. The complexation of methotrexate within a dendrimer changes the water insoluble drug into a stable and readily water-soluble compound. When this dendrimer complexed drug, however, was placed in a solution of phosphate buffered saline, the methotrexate was immediately released and displayed diffusion characteristics identical to free methotrexate. Covalently coupled methotrexate dendrimer conjugates were stable under identical conditions in water and buffered saline. Cytotoxicity tests showed that methotrexate as the dendrimer inclusion complex had an activity identical to the free drug in vitro. In contrast, folic acid targeted dendrimer with covalently conjugated methotrexate specifically killed receptor-expressing cells by intracellular delivery of the drug through receptor-mediated endocytosis. This study demonstrates that while drug as a dendrimer inclusion complex is readily released and active in vitro, covalently conjugated drug to dendrimer is better suited for specifically targeted drug delivery.

Cell Survival↗
PubMed2005

Lipid bilayer disruption by polycationic polymers: the roles of size and chemical functional group.

Polycationic polymers are used extensively in biology to disrupt cell membranes and thus enhance the transport of materials into the cell. The highly polydisperse nature of many of these materials makes obtaining a mechanistic understanding of the disruption processes difficult. To design an effective mechanistic study, a monodisperse class of polycationic polymers, poly(amidoamine) (PAMAM) dendrimers, has been studied in the context of supported dimyristoylphosphatidylcholine (DMPC) lipid bilayers using atomic force microscopy (AFM). Aqueous solutions of amine-terminated generation 7 (G7) PAMAM dendrimers caused the formation of 15-40-nm-diameter holes in lipid bilayers. This effect was significantly reduced for smaller G5 dendrimers. For G3, no hole formation was observed. In addition to dendrimer size, surface chemistry had a strong influence on dendrimer-lipid bilayer interactions. In particular, acetamide-terminated G5 did not cause hole formation in bilayers. In all instances, the edges of bilayer defects proved to be points of highest dendrimer activity. A proposed mechanism for the removal of lipids by dendrimers involves the formation of dendrimer-filled lipid vesicles. By considering the thermodynamics, interaction free energy, and geometry of these self-assembled vesicles, a model that explains the influence of polymer particle size and surface chemistry on the interactions with lipid membranes was developed. These results are of general significance for understanding the physical and chemical properties of polycationic polymer interactions with membranes that lead to the transport of materials across cell membranes.

Cations↗
PubMed2005

Poly(amidoamine) dendrimer-based multifunctional engineered nanodevice for cancer therapy.

Multifunctional cancer therapeutic nanodevices have been designed and synthesized using the poly(amidoamine) (PAMAM) dendrimer as a carrier. Partial acetylation of the generation 5 (G5) PAMAM dendrimer was utilized to neutralize a fraction of the primary amino groups, provide enhanced solubility of the dendrimer during the conjugation reaction of fluorescein isothiocyanate (FITC) (in dimethyl sulfoxide (DMSO)), and prevent nonspecific targeting interactions (in vitro and in vivo) during delivery. The remaining nonacetylated primary amino groups were utilized for conjugation of the functional molecules fluorescein isothiocyanate (FITC, an imaging agent), folic acid (FA, targets overexpressed folate receptors on specific cancer cells), and methotrexate (MTX, chemotherapeutic drug). The appropriate control nanodevices have been synthesized as well. The G5 PAMAM dendrimer molecular weight and number of primary amino groups were determined by gel permeation chromatography (GPC) and potentiometric titration for stoichiometric design of ensuing conjugation reactions. Additionally, dendrimer conjugates were characterized by multiple analytical methods including GPC, nuclear magnetic resonance spectroscopy (NMR), high performance liquid chromatography (HPLC), and UV spectroscopy. The fully characterized nanodevices can be used for the targeted delivery of chemotherapeutic and imaging agents to specific cancer cells. Here, we present a more extensive investigation of our previously reported synthesis of this material with improvements directed toward scale-up synthesis and clinical trials (Pharm. Res. 2002, 19 (9), 1310-1316).

Acetylation↗
PubMed2005

Death ligand tumor necrosis factor-related apoptosis-inducing ligand inhibits experimental autoimmune thyroiditis.

The role of TNF-related apoptosis-inducing ligand (TRAIL) in autoimmune thyroiditis is unclear. We used experimental autoimmune thyroiditis to clarify the contribution of TRAIL to the development of autoimmune thyroiditis. CBA/J mice were immunized with murine thyroglobulin, and spleen cells from these mice were subsequently injected into irradiated recipient CBA/J mice. One week later, the recipient mice were treated with recombinant TRAIL or a control protein. Compared with control animals, TRAIL-treated mice developed a milder form of the disease with a significant decrease in mononuclear cell infiltration in the thyroid and less thyroid follicular destruction. Furthermore, the number of apoptotic thyrocytes and also thyroglobulin-specific T helper-1 cell responses in TRAIL-treated mice was lower than that in the control animals. This study suggests that exogenous TRAIL suppresses the development of autoimmune thyroiditis via altering the function of cells involved in the immune response. These findings may contribute toward a novel treatment autoimmune thyroiditis.

Animals↗
PubMed2005

HPLC analysis of PAMAM dendrimer based multifunctional devices.

Comprehensive high-performance liquid chromatography (HPLC) analyses were performed on poly(amidoamine) (PAMAM) dendrimer based multifunctional devices. The nanometer-size devices were synthesized by conjugating partially acetylated (Ac) poly(amidoamine) dendrimers of generation 5 (G5) with fluorescein isothiocyanate (FITC), folic acid (FA) and methotrexate (MTX). The devices are intended for targeted intracellular drug delivery to tumor cells through the folate receptor. Methods were developed for detection and separation of various surface functionalized dendrimer conjugates and small molecules (FITC, FA, MTX) using a common gradient. Results indicate that the HPLC technique can be used as a quality control tool for determining purity of the G5 carrier, its acetylated form, and mono-, bi- and tri-functional nanodevices. More importantly, the chromatograms of these novel nanodevices, reported for the first time, provide information on critical properties such as polydispersity, surface heterogeneity and solubility. The benchmark data can be used to optimize the physicochemical properties of the conjugates to improve drug delivery to cancer cells.

Chromatography, High Pressure Liquid↗
PubMed2005

Nanoparticle targeting of anticancer drug improves therapeutic response in animal model of human epithelial cancer.

Prior studies suggested that nanoparticle drug delivery might improve the therapeutic response to anticancer drugs and allow the simultaneous monitoring of drug uptake by tumors. We employed modified PAMAM dendritic polymers <5 nm in diameter as carriers. Acetylated dendrimers were conjugated to folic acid as a targeting agent and then coupled to either methotrexate or tritium and either fluorescein or 6-carboxytetramethylrhodamine. These conjugates were injected i.v. into immunodeficient mice bearing human KB tumors that overexpress the folic acid receptor. In contrast to nontargeted polymer, folate-conjugated nanoparticles concentrated in the tumor and liver tissue over 4 days after administration. The tumor tissue localization of the folate-targeted polymer could be attenuated by prior i.v. injection of free folic acid. Confocal microscopy confirmed the internalization of the drug conjugates into the tumor cells. Targeting methotrexate increased its antitumor activity and markedly decreased its toxicity, allowing therapeutic responses not possible with a free drug.

Animals↗