Publication Archives

2003

Specific Association of Thiamine-Coated Gadolinium Nanoparticles with Human Breast Cancer Cells Expressing Thiamine Transporters.

MO Oyewumi, S Liu, JA Moscow, and RJ Mumper. S

Bioconj. Chem. (2003) 14:404-411.

Division of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, Lexington 40536-0082, USA.

Thiamine (vitamin B(1)) was investigated as a tumor-specific ligand for gadolinium nanoparticles. Solid nanoparticles containing gadolinium hexanedione (1.5 mg/mL) were engineered from oil-in-water microemulsion templates and coated with thiamine ligands. Thiamine ligands were synthesized by conjugating thiamine to either distearoylphosphatidylethanolamine (DSPE) or fluorescein via a poly(ethylene glycol) (PEG) spacer (Mw 3350). The efficiency of thiamine ligand attachment to nanoparticles was evaluated using gel permeation chromatography (GPC). Cell association studies were carried using a methotrexate-resistant breast cancer cell line, MTX(R)ZR75, transfected with thiamine transporter genes (THTR1 and THTR2). Thiamine-coated nanoparticle association with THTR1 and THTR2 cells was significantly greater than that with control breast cancer cells (MTX(R)ZR75 transfected with the empty expression vector pREP4) (p < 0.01; t-test). The nanoparticle cell association was significantly dependent on the extent of thiamine ligand coating on nanoparticles, expression of thiamine transporters in cells, temperature of incubation, and the concentration of competitive inhibitor (free thiamine). Further studies are warranted to assess the potential of the engineered thiamine-coated gadolinium (Gd) nanoparticles in neutron capture therapy of tumors.

PMID: 12643751 [PubMed - indexed for MEDLINE]

Preparation and Characterization of Novel Coenzyme Q10 Nanoparticles Engineered from Microemulsion Precursors.

Hsu CH, Cui Z, Mumper RJ, Jay M.

AAPS PharmSciTech. 2003;4(3):E32.

Division of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, Lexington, KY 40536-0082, USA.

AAPS PharmSciTech. 2003;4(3):E32.

The purpose of these studies was to prepare and characterize nanoparticles into which Coenzyme Q10 (CoQ10) had been incorporated (CoQ10-NPs) using a simple and potentially scalable method. CoQ10-NPs were prepared by cooling warm microemulsion precursors composed of emulsifying wax, CoQ10, Brij 78, and/or Tween 20. The nanoparticles were lyophilized, and the stability of CoQ10-NPs in both lyophilized form and aqueous suspension was monitored over 7 days. The release of CoQ10 from the nanoparticles was investigated at 37 degrees C. Finally, an in vitro study of the uptake of CoQ10-NPs by mouse macrophage, J774A.1, was completed. The incorporation efficiency of CoQ10 was approximately 74% +/- 5%. Differential Scanning Calorimetry (DSC) showed that the nanoparticle was not a physical mixture of its individual components. The size of the nanoparticles increased over time if stored in aqueous suspension. However, enhanced stability was observed when the nanoparticles were stored at 4 degrees C. Storage in lyophilized form demonstrated the highest stability. The in vitro release profile of CoQ10 from the nanoparticles showed an initial period of rapid release in the first 9 hours followed by a period of slower and extended release. The uptake of CoQ10-NPs by the J774A.1 cells was over 4-fold higher than that of the CoQ10-free nanoparticles (P < .05). In conclusion, CoQ10-NPs with potential application for oral CoQ10 delivery were engineered readily from microemulsion precursors.

PMID: 14621964 [PubMed - indexed for MEDLINE]

Influence of Formulation Parameters on Gadolinium Entrapment and Tumor Cell Uptake Using Folate-Coated Nanoparticles

MO Oyewumi and RJ Mumper.

Inter. J. Pharm. (2003) 251:85-97.

Division of Pharmaceutical Sciences, Center for Pharmaceutical Science and Technology, College of Pharmacy, University of Kentucky, 907 Rose Street, Lexington, KY 40536-0082, USA.

Emulsifying wax and polyoxyl 2 stearyl ether (Brij 72) nanoparticles (2 mg/ml) containing high concentrations of gadolinium hexanedione (GdH) (0-3 mg) have been engineered from oil-in-water microemulsion templates. Solid nanoparticles were cured by cooling warm microemulsion templates (prepared at 55 degrees C) to room temperature in one vessel. Nanoparticles were characterized by transmission electron microscopy (TEM), photon correlation spectroscopy (PCS) and gel permeation chromatography (GPC). To obtain folate-coated nanoparticles, a folate ligand was added to either the microemulsion templates or nanoparticle suspensions at 25 degrees C. Since the concentration of Gd in the tumor is critical to the success of Gd-neutron capture therapy (NCT), the effects of various formulation factors on GdH entrapment in nanoparticles as well as tumor-targeting were studied. GdH entrapment in nanoparticles was affected mostly by the method of GdH incorporation and surfactant concentration used in preparing the microemulsion templates. Cell uptake studies were carried out in KB cells (human nasopharyngeal epidermal carcinoma cell line). The method of adding folate ligand to the formulations did not significantly affect nanoparticle cell uptake (P>0.11; t-test). However, the concentration of folate ligand added to nanoparticles had the greatest influence on nanoparticle uptake (P<0.01; t-test). The results showed that GdH entrapment and cell uptake were optimized and suggested that engineered folate-coated nanoparticles may serve as effective carrier systems for Gd-NCT of tumors.

PMID: 12527178 [PubMed - indexed for MEDLINE]

Brain Uptake of Thiamine-Coated Nanoparticles

PR Lockman, MO Oyewumi, JM Koziara, KE Roder, RJ Mumper, and DD Allen.

J. Controlled Rel. (2003) 93:271-282.

Department of Pharmaceutical Sciences, School of Pharmacy, Texas Tech University HSC, Amarillo, TX 79106-1712, USA.

Recently, a novel nanoparticle (NP) comprised of emulsifying wax and Brij 78 was shown to have significant brain uptake using the in-situ rat brain perfusion technique. To further these studies and to specifically target brain, we have incorporated thiamine as a surface ligand on the nanoparticles. Solid nanoparticles were prepared from oil-in-water microemulsion precursors. Nanoparticles were radiolabeled and a thiamine ligand (thiamine linked to distearoylphosphatidylethanolamine via a polyethylene glycol spacer) was coated on the surface of the nanoparticles. Initial experiments focused on assessing uptake of [3H]nanoparticles with and without thiamine surface ligands. Biodistribution nanoparticle studies were also carried out in BALB/c mice. The results showed: (1) the effectiveness of using microemulsions as precursors to engineer nanoparticles, (2) kinetic modeling for brain uptake of nanoparticles with and without the thiamine surface ligands, and (3) initial data suggesting mechanisms for nanoparticle brain entry. Comparison of NP brain uptake demonstrated that the thiamine-coated nanoparticle associated with the blood-brain barrier (BBB) thiamine transporter and had an increased K(in) between 45 and 120 s (thiamine coated NP 9.8 +/- 1.1 x 10(-3) ml/s/g versus uncoated NPs; 7.0 +/- 0.3 x 10(-3) ml/s/g). It was concluded that the thiamine ligand facilitated binding and/or association with blood-brain barrier thiamine transporters, which may be a viable mechanism for nanoparticle mediated brain drug delivery.

PMID: 14644577 [PubMed - indexed for MEDLINE]

In-Situ Blood-Brain Barrier Transport of Nanoparticles

JM Koziara, PR Lockman, DD Allen, and RJ Mumper.

Pharm. Res. (2003) 20:1772-1778.

Division of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, Lexington, KY 40536-0082, USA.

PURPOSE: Two novel types of nanoparticles were evaluated as poten tial carriers for drugs across the blood-brain barrier (BBB). METHODS: Nanoparticles were composed of biocompatible materials including emulsifying wax (E. Wax) or Brij 72. Brij 78 and Tween 80 were used as surfactants for E. Wax nanoparticles (E78 NPs) and Brij 72 nanoparticles (E72 NPs), respectively. Both nanoparticle formulations were prepared from warm microemulsion precursors usin melted E. Wax or Brij 72 as the oil phase. Nanoparticles were radio-labeled by entrapment of [3H]cetyl alcohol, and entrapment efficiency and release of radiolabel were evaluated. The transport of E78 and E72 NPs across the BBB was measured by an in situ rat brai perfusion method. RESULTS: Both formulations were successfully radiolabeled by entrapment of [3H]cetyl alcohol; -98% of radiolabel remained associated with nanoparticles at experimental conditions. The transfer rate (Kin) of E78 NPs from perfusion fluid into the brain was 4.1 +/- 0.5 x 10(-3) ml/s/g, and the permeability-surface area product (PA) was 4.3 +/- 0.7 x 10(-3) ml/s/g. The values for Kin and PA for E72 NPs were 5.7 +/- 1.1 x 10(-3) ml/s/g and 6.1 +/- 1.4 x 10(-3) ml/s/g, respectively. CONCLUSIONS: For both nanoparticle types, statistically significant uptake was observed compared to [14C]sucrose, suggesting central nervous system uptake of nanoparticles. The mechanism underlying th nanoparticle brain uptake has yet to be fully understood.

PMID: 14661921 [PubMed - indexed for MEDLINE]

Evaluation of Blood-Brain Barrier Thiamine Efflux Using the In-Situ Rat Brain Perfusion Methods

PR Lockman, RJ Mumper, and DD Allen.

J. Neurochemistry. (2003) 86:627-634.

Department of Pharmaceutical Sciences, School of Pharmacy, Texas Tech University HSC, Amarillo, Texas 79106-1712, USA.

Thiamine is an essential, positively charged (under physiologic conditions), water-soluble vitamin requiring transport into brain. Brain thiamine deficiency has been linked to neurodegenerative disease by subsequent impairment of thiamine-dependent enzymes used in brain glucose/energy metabolism. In this report, we evaluate brain uptake and efflux of [3H]thiamine using the in situ rat brain perfusion technique. To confirm brain distribution was not related to blood-brain barrier endothelial cell uptake, we compared parenchymal and cell distribution of [3H]thiamine using capillary depletion. Our work supports previous literature findings suggesting blood-brain barrier thiamine uptake is via a carrier-mediated transport mechanism, yet extends the literature by redefining the kinetics with more sensitive methodology. Significantly, [3H]thiamine brain accumulation was influenced by a considerable efflux rate. Evaluation of the efflux mechanism demonstrated increased stimulation by the presence of increased vascular thiamine. The influx transport mechanism and efflux rate were each comparable throughout brain regions despite documented differences in glucose and thiamine metabolism. The observation that [3H]thiamine blood-brain barrier influx and efflux is regionally homogenous may have significant relevance to neurodegenerative disease linked to thiamine deficiency.

PMID: 12859676 [PubMed - indexed for MEDLINE]

Genetic Immunization by Jet Injection of Targeted pDNA-Coated Nanoparticles

RJ Mumper and Z Cui.

Pharm Res. (2003) 31:255-262.

Division of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, 907 Rose Street, Lexington, KY 40536-0082, USA. rjmump2@uky.edu

Genetic immunization strategies have largely focused on the use of "naked" plasmid DNA or the gene gun. However, there remains a clear need to further improve the efficiency and/or cost of potential DNA vaccines. The theoretical basis of our research is to rationally design genetic immunization methodologies for nanoparticle-based delivery systems of plasmid DNA, perhaps in combination with already commercially available needle-free devices, such as the Biojector 2000. These methodologies may both reduce the dose of pDNA required and enhance the breadth and depth of protective immune responses (i.e., humoral and cellular). The purpose of this article is to provide detailed experimental methods to (1) engineer and characterize pDNA-coated cationic nanoparticles (<100nm) directly from oil-in-water microemulsion precursors and (2) enhance both the breadth and depth of immune responses after immunization of mice with pDNA-coated nanoparticles by different routes of administration, including intradermal, using a needle-free jet injection device.

PMID: 14511958 [PubMed - indexed for MEDLINE]

Novel Ethanol-in-Fluorocarbon Microemulsions for Topical Genetic Immunization

Z Cui, W Fountain, M Clark, M Jay, and RJ Mumper.

Pharm. Res. (2003) 20:16-23.

Division of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, Lexington, Kentucky 40536-0082, USA.

PURPOSE: Traditionally, vaccines have been administered by needle injection. Topical immunization through the intact skin with either protein- or DNA-based vaccines has attracted much attention recently. We sought to enhance the immune responses induced by DNA-based vaccines after topical application by developing novel ethanol-in-fluorocarbon (E/F) microemulsion systems to aid in the delivery of plasmid DNA (pDNA). METHODS: Ten different fluorosurfactants were selected or synthesized and screened by pseudo-phase-diagram construction for their ability to form E/F microemulsions. Plasmid DNA was successfully incorporated into E/F microemulsions using several different fluorosurfactants and perfluorooctyl bromide as the continuous fluorocarbon phase. For several reasons, Zonyl FSN-100 (an ethoxylated nonionic fluorosurfactant) was selected for further studies. In vivo studies were performed in mice to assess pDNA expression in skin and immunologic responses after topical application of this system using a luciferase-encoding plasmid (CMV-luciferase) and a CMV-beta-galactosidase-encoding plasmid, respectively. RESULTS: Plasmid DNA incorporated into E/F microemulsion using FSN-100 as the surfactant was found to be stable. After topical application of this E/F microemulsion system, significant enhancements in luciferase expression and antibody and T-helper type-1 biased immune responses were observed relative to those of "naked" pDNA in saline or ethanol. For example, with the E/F microemulsion system, the specific serum IgG and IgA titers were increased by 45-fold and over 1000-fold, respectively. CONCLUSION: A novel fluorocarbon-based microemulsion system for potential DNA vaccine delivery was developed.

PMID: 12608531 [PubMed - indexed for MEDLINE]

Microparticles and Nanoparticles as Delivery Systems for DNA Vaccines

Z Cui and RJ Mumper.

Crit. Rev. Ther. Drug Carrier Sys. (2003) 20(2&3):103-137.

Center for Pharmaceutical Science and Technology, College of Pharmacy, University of Kentucky, Lexington, KY 40536, USA.

DNA vaccines, also referred to as genetic vaccines, are generating significant preclinical and clinical interest. It has been proven that the expression of an antigen or antigens from plasmid DNA (pDNA) may elicit both humoral and cellular immune responses. Therefore, DNA vaccines may have potential as new vaccines for important pathogens such as HIV, hepatitis C, tuberculosis, and malaria. However, the clinical results using "naked" pDNA have been disappointing in the breadth and depth of the immune response and the relatively high doses of pDNA needed to elicit a response. Clinical trials with the gene gun have been promising, but it is unclear whether this technology will be commercially viable. As a result, there exists a clear need for new vaccine delivery systems that can be administered at low doses to elicit strong humoral and cellular immune responses. One promising approach is the development of microparticles and nanoparticles as delivery systems for DNA vaccines. In this review, the application of microparticles and nanoparticles as DNA vaccine delivery systems will be critically reviewed with a primary focus on those systems that have generated in vivo immune responses.

PMID: 14584521 [PubMed - indexed for MEDLINE]

Physical Characterization and Macrophage Cell Uptake of Mannan-Coated Nanoparticles

Z Cui, CH Hsu, and RJ Mumper.

Drug Dev. Ind. Pharm. (2003) 29:689-700.

Division of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, Lexington, Kentucky 40536-0082, USA.

Previously, we reported on a cationic nanoparticle-based DNA vaccine delivery system engineered from warm oil-in-water microemulsion precursors. In these present studies, the feasibility of lyophilizing the nanoparticles and their thermal properties were investigated. Also, the binding and uptake of the nanoparticles by a macrophage cell line were studied. The nanoparticles (prior to pDNA coating) were freeze-dried with lactose or sucrose as cryoprotectants. The stability of lyophilized nanoparticles at room temperature was monitored and compared to that of the aqueous nanoparticle suspension. The thermal properties of the nanoparticles were investigated using differential scanning calorimetry (DSC). The nanoparticles, coated or uncoated with mannan as a ligand, were incubated with a mannose receptor positive (MR+) mouse macrophage cell line (J774E), at either 4 degrees C or 37 degrees C to study the binding and uptake of the nanoparticles by the cells. It was found that lactose or sucrose (1-5%, w/v) was required for successful lyophilization of the nanoparticles. After 4 months of storage, the size of lyophilized nanoparticles did not significantly increase while those in aqueous suspension grew by over 900%. Unlike its individual components, emulsifying wax (m.p., approximately 55 degrees C) and hexadecyltrimethyl ammonium bromide, the nanoparticles showed a melting point of approximately 90 degrees C. Moreover, the DSC profile of the nanoparticles was different from that of the physical mixture of emulsifying wax and CTAB. After 1 hour incubation at 37 degrees C, the uptake of mannan-coated nanoparticles was 50% higher than that of the uncoated nanoparticles. At 4 degrees C and after one hour, the binding of the mannan-coated nanoparticles by J774E was over 2-fold higher than that of the uncoated nanoparticles. This increase in J774E binding could be abolished by preincubating the cells with free mannan, suggesting that the binding and uptake were receptor-mediated. In conclusion, the nanoparticles were lyophilizable, and lyophilization was shown to enhance the stability of the nanoparticles. DSC provided evidence that the nanoparticles were not a physical mixture of their individual components. Finally, cell binding and uptake studies demonstrated that the nanoparticles have potential application for cell-specific targeting.

PMID: 12889787 [PubMed - indexed for MEDLINE]

Near-Infrared Spectroscopy for the Determination of Testosterone in Thin-Film Composites.

W. Fountain, K Dumstorf, A.E. Lowell, R Lodder, and RJ Mumper.

J Pharm Biomed Anal. (2003) Sep 19;33(2):181-9.

Division of Pharmaceutical Sciences, Center for Pharmaceutical Science and Technology, College of Pharmacy, University of Kentucky, Lexington, KY 40536-0082, USA.

More rapid, reproducible, and cost-effective methods to control product quality in the pharmaceutical industry continue to be a major emphasis, particularly with the FDA through its recent process analytical technologies (PAT) initiative. Many different methods have been used to determine the stability and content uniformity of a drug in various dosage forms; however, most of these methods include the destruction of the sample. Therefore, the development of nondestructive methods that allow the analysis of each individual dosage form has become the basis of much research. A new assay for the nondestructive determination of testosterone content in mucoadhesive bi-layer thin-film composites (TFCs) using near-infrared spectroscopy (NIR) was developed. Five sets of the circular films (n=5) with theoretical testosterone content of 0, 1, 2, 3, and 4 mg per 3/8th in. diameter disks were scanned in the near-infrared region of 1100-2500 nm to determine testosterone content. The NIR results were directly compared with those obtained using a previously developed ultraviolet assay for testosterone at 240 nm. Principal component regression (PCR) was performed to calibrate the NIR assay. This correlation produced r2=0.99 with a standard error of estimate (SEE)=0.18 mg, and a standard error of performance (SEP)=0.18 on cross validation with an equal number of samples (F test passed at P=0.05). Though the UV assay showed a slightly better r2 value, the NIR assay was much quicker, easier, and nondestructive. Therefore, the NIR assay may have significant potential for use in the quality control of pharmaceutical films containing drugs.

PMID: 12972083 [PubMed - indexed for MEDLINE]

In Vivo and In Vitro Assessment of Baseline Blood-Brain Barrier Parameters in the Presence of Novel Nanoparticles

Lockman PR, Koziara J, Roder KE, Paulson J, Abbruscato TJ, Mumper RJ, Allen DD.

Pharm Res. 2003 May;20(5):705-13.

Department of Pharmaceutical Sciences, School of Pharmacy, Texas Tech University HSC, 1300 So. Coulter Dr., Amarillo, Texas 79106-1712, USA.

PURPOSE: Nanoparticles have advantage as CNS drug delivery vehicles given they disguise drug permeation limiting characteristics. Conflicting toxicological data, however, is published with regard to blood-brain barrier integrity and gross mortality. METHODS: To address this issue two novel nanoparticle types: "emulsifying wax/Brij 78" and "Brij 72/Tween 80 nanoparticles were evaluated in vivo for effect on cerebral perfusion flow, barrier integrity, and permeability using the in situ brain perfusion technique. Additional evaluation was completed in vitro using bovine brain microvessel endothelial cells for effect on integrity, permeability, cationic transport interactions, and tight junction protein expression. RESULTS: In the presence of either nanoparticle formulation, no overall significant differences were observed for cerebral perfusion flow in vivo. Furthermore, observed in vitro and in vivo data showed no statistical changes in barrier integrity, membrane permeability, or facilitated choline transport. Western blot analyses of occludin and claudin-1 confirmed no protein expression changes with incubation of either nanoparticle. CONCLUSIONS: The nanoparticle formulations appear to have no effect on primary BBB parameters in established in vitro and in vivo blood-brain barrier models.

Publication Types:
Comparative Study
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, P.H.S.

PMID: 12751624 [PubMed - indexed for MEDLINE]

Intradermal Immunization with Novel Plasmid DNA-Coated Nanoparticles via a Needle-Free Injection Device

Cui Z, Baizer L, Mumper RJ.

J Biotechnol. 2003 Apr 24;102(2):105-15.

Division of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky,Lexington, KY 40536-0082, USA.

A high population of dendritic cells in the skin makes intradermal (ID) immunization an attractive route. We sought to further enhance immune responses from a previously reported novel nanoparticle-based DNA vaccine delivery system by administering the system intradermally into mouse skin using Biojector 2000, a needle-free jet injection device. Two mouse studies were carried out. Balb/C mice (n=5-6) were immunized on day 0, 7, and 14 by subcutaneous injection or via the Biojector 2000 with pDNA alone (CMV-beta-galactosidase, 5 micro g), pDNA-coated nanoparticles, or beta-galactosidase protein (10 micro g) adjuvanted with 'Alum' (15 micro g). On day 28, mice were sacrificed and specific serum IgG and IgA titer, in vitro cytokine release, and cell proliferation of isolated splenocytes were determined. Similar to previous reports, in both mouse studies, SC immunization with pDNA-coated nanoparticles led to over a log increase in specific serum IgG titer as compared to immunization with pDNA alone. For pDNA alone, jet and SC injection did not result in significant differences in IgG titer. In contrast, for pDNA-coated nanoparticles, jet injection led to as high as a 20-fold enhancement in IgG titer over SC injection. In addition, jet injection of pDNA-coated nanoparticles enhanced the IgG titer by more than 200-fold over jet injection of pDNA alone. Also, jet injection of pDNA-coated nanoparticles resulted in significantly enhanced specific serum IgA titer. For in vitro cytokine release, immunization with pDNA-coated nanoparticles by jet injection enhanced IFN-gamma and IL-4 release over pDNA alone by 6- and 5-fold,respectively. SC injection of pDNA-coated nanoparticles also resulted in enhanced IFN-gamma and IL-4 release over pDNA alone although with less magnitude. Finally,immunization with pDNA-coated nanoparticles, by both jet injection and SC injection, led to improved splenocyte proliferation over pDNA alone. In conclusion, a combination of a novel cationic nanoparticle-based DNA delivery system with ID jet injection led to enhanced antibody production, Th-1/Th-2 balanced cytokine release, and enhanced splenocyte proliferation.

Publication Types:
Comparative Study

PMID: 12697387 [PubMed - indexed for MEDLINE]

The Effect of Co-Administration fo Adjuvants with a Nanoparticle-Based Genetic Vaccine Delivery System on the Resulting Immune Responses

Cui Z, Mumper RJ.

Eur J Pharm Biopharm. 2003 Jan;55(1):11-8.

Division of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky,Lexington, KY 40536, USA.

Previously, we reported on a novel cationic nanoparticle-based DNA vaccine delivery system. In the present studies, the effects of co-administration of two well-known adjuvants, cholera toxin (CT) and lipid A (LA), with plasmid DNA (pDNA)-coated nanoparticles were investigated. Balb/C mice (n=6) were immunized with either pDNA alone cytomegalovirus-beta-galactosidase, 5 microg) or pDNA-coated nanoparticles with either 0 or 50 microg of LA on days 0, 7, and 14 subcutaneously (s.c.), or topically on shaved skin with either pDNA (5 microg) alone or pDNA-coated nanoparticles with 0, 10, or 100 microg of CT on days 0, 6, 21, and 35. Mice were sacrificed on day 28 or day 45. Serum IgG titer, in vitro cytokine release and cell proliferation of the isolated splenocytes were determined. By the topical route, immunization of mice with 'naked' pDNA together with 10 and 100 microg of CT significantly enhanced the antigen-specific serum IgG titer by four- and 20-fold, respectively, compared to immunization with pDNA alone. Moreover, co-administration of 100 microg CT with the pDNA-nanoparticles enhanced the IgG titer by more than 300-fold over immunization with 'naked' pDNA alone with no CT. In vitro interferon-gamma (IFN)-gamma release from splenocytes isolated from mice immunized with pDNA-coated nanoparticles with CT (100 microg) was increased by three-fold over immunization with pDNA-nanoparticles without CT. Similarly, in vitro IFN-gamma release from splenocytes isolated from mice immunized with 'naked' pDNA with CT (100 microg) was increased by two-fold over immunization with 'naked' pDNA without CT. Finally, pDNA-coated nanoparticles adjuvanted with 10 microg CT resulted in the strongest splenocyte proliferation. By the s.c. route, co-administration of LA (50 microg) with pDNA resulted in more than 16-fold enhancement in IgG titer over immunization with 'naked' pDNA alone. Immunization with pDNA-coated nanoparticles with LA (50 microg) led to 16-fold enhancement in specific serum IgG titer over immunization with pDNA-coated nanoparticles with no LA, and more than 250-fold enhancement over immunization with 'naked' pDNA alone with no LA. Moreover, in vitro IFN-gamma release and proliferation by splenocytes isolated from LA co-immunized mice was also significantly enhanced. In conclusion, CT (topical) and LA (s.c.) are potential adjuvants to further enhance immune responses using a novel cationic nanoparticle-based DNA vaccine delivery system.

PMID: 12551699 [PubMed - indexed for MEDLINE]