Biomedical subjects

Zhengrong Cui

Publications and source records attributed to Zhengrong Cui.

12 recordsLinked to original sources

PubMed2003

Intradermal immunization with novel plasmid DNA-coated nanoparticles via a needle-free injection device.

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.

Animals↗

PubMed2003

The effect of co-administration of adjuvants with a nanoparticle-based genetic vaccine delivery system on the resulting immune responses.

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.

Adjuvants, Immunologic↗

PubMed2003

Novel ethanol-in-fluorocarbon microemulsions for topical genetic immunization.

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.

Administration, Topical↗

PubMed2003

Physical characterization and macrophage cell uptake of mannan-coated nanoparticles.

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.

Animals↗

PubMed2002

Topical immunization using nanoengineered genetic vaccines.

DNA vaccines have been shown to elicit both broad humoral and cellular immune responses. Needle-free injection devices and the gene gun have been used to deliver these DNA vaccines to dendritic cells in the viable skin epidermis with some success. However, more cost-effective and dendritic cell (DC)-targeted immunization strategies are sought. To this end, a nanoengineered genetic vaccine for simple topical application was developed. Expressed beta-galactosidase was used as a model antigen. Plasmid DNA was coated on the surface of preformed cationic nanoparticles engineered directly from warm oil-in-water (O/W) microemulsion precursors comprised of emulsifying wax as the oil phase and CTAB as a cationic surfactant. Mannan, a DC ligand, was coated on the nanoparticles with and without entrapped endosomolytic agents, dioleoyl phosphatidylethanolamine (DOPE) and cholesterol. In-vitro cell transfection studies were performed to confirm transgene expression with these pDNA-coated nanoparticles. An in-vitro Concanavalin A (ConA) agglutination assay confirmed the presence of mannan on the surface of nanoparticles. The humoral and proliferative immune responses were assessed after topical application of these nanoengineered systems to the skin of shaved Balb/C mice. All pDNA-coated nanoparticles, especially the mannan-coated pDNA-nanoparticles with DOPE, resulted in significant enhancement in both antigen-specific IgG titers (16-fold) and splenocyte proliferation over 'naked' pDNA alone.

Administration, Topical↗

PubMed2002

Coating of cationized protein on engineered nanoparticles results in enhanced immune responses.

A significant emphasis has been placed on the development of improved adjuvants and delivery systems to improve both antibody production and cell-mediated immunity. The overall goal of this project was to cationize a model protein antigen, beta-galactosidase (nGal), coat the cationized Gal (cGal) on the surface of novel anionic nanoparticles engineered from microemulsion precursors, and assess the immune response of this system after subcutaneous injection to mice. Gal was chemically cationized as evidenced by gel electrophoresis. The cGal was coated on anionic nanoparticles (78+/-11 nm) engineered from oil-in-water microemulsion precursors to produce cGal-coated nanoparticles. The immune response to nGal with 'Alum', cGal alone, and cGal-coated nanoparticles were assessed after subcutaneous injection to Balb/c mice. cGal alone elicited similar antibody titer to nGal with 'Alum'. However, cGal-coated nanoparticles elicited the strongest and most reproducible antibody titer. cGal alone produced very high levels of Th1 cytokines, but low levels of Th2 cytokines. In contrast, cGal-coated nanoparticles significantly enhanced both the Th1 and Th2 cytokines. The results demonstrated the utility of antigen-coated nanoparticles to enhance both the humoral and Th1-type immune responses, in parallel.

Adjuvants, Immunologic↗

PubMed2002

Transmucosal delivery of testosterone in rabbits using novel bi-layer mucoadhesive wax-film composite disks.

Testosterone exhibits very low oral bioavailability because of its low aqueous solubility and extensive first-pass metabolism. The purpose of this study was to develop a novel bi-layer mucoadhesive wax-film composite (WFC), and to test the relative bioavailability of testosterone via the buccal route in rabbits. The release rate of testosterone from optimal WFCs (3/8-in. diameter) per unit surface area was 5.6 microg x cm(2) x mL(-1) x min(-1) and was zero-order. Bi-layer WFCs (average weight of 14 +/- 2.6 mg and thickness of 186 +/- 34 microns) containing 4 mg of testosterone were applied to the buccal pouch of anesthetized New Zealand white rabbits. Rabbits (n = 3) injected intravenously had C(max) and area under the curve values of 1200 +/- 46 ng/mL, and 48,227 +/- 12,995 ng x min/mL, respectively. Rabbits (n = 3) dosed via the buccal pouch had C(max), T(max), and area under the curve values of 127 +/- 13 ng/mL, 200 +/- 35 min, and 24,221 +/- 1543 ng x min/mL. The relative bioavailability for rabbits treated with the WFC was 50.2 +/- 3.2% with a coefficient of variation of 6.4%. It was concluded that these bi-layer mucoadhesive WFCs disks could deliver physiologically relevant amounts of insoluble drugs such as testosterone across the buccal mucosa.

Acrylic Resins↗

PubMed2002

Genetic immunization using nanoparticles engineered from microemulsion precursors.

PURPOSE: Genetic immunization using "naked" plasmid DNA (pDNA) has been shown to elicit broad humoral and cellular immune responses. However, more versatile and perhaps cell-targeted delivery systems are needed. To this end, a novel process to engineer cationic nanoparticles coated with pDNA for genetic immunization was explored. METHODS; Cationic nanoparticles were engineered from warm oil-in-water microemulsion precursors composed of emulsifying wax as the oil phase and cetyltrimethylammonium bromide (CTAB) as the cationic surfactant. Plasmid DNA was coated on the surface of the cationic nanoparticles to produce pDNA-coated nanoparticles. An endosomolytic lipid and/or a dendritic cell-targeting ligand (mannan) were incorporated in or deposited on the nanoparticles to enhance the in vitro cell transfection efficiency and the in vivo immune responses after subcutaneous injection to Balb/C mice. The IgG titer to expressed beta-galactosidase and the cytokine release from isolated splenocytes after stimulation were determined on 28 days. RESULTS: Cationic nanoparticles (around 100 nm) were engineered within minutes. The pDNA-coated nanoparticles were stable at 37 degrees C over 30 min in selected biologic fluids. Transmission electron microscopy showed the nanoparticles were spherical. Plasmid DNA-coated nanoparticles. especially those with both an endosomolytic lipid and dendritic cell-targeting ligand. resulted in significant enhancement in both IgG titer (over 16-fold) and T-helper type-1 (Th1-type) cytokine release (up to 300% increase) over "naked" pDNA. CONCLUSION: A novel method to engineer pDNA-coated nanoparticles for enhanced in vitro cell transfection and enhanced in vivo immune responses was reported.

Animals↗

PubMed2002

Bilayer films for mucosal (genetic) immunization via the buccal route in rabbits.

PURPOSE: The oral buccal mucosa may be an ideal site for mucosal immunization, allowing for the needle-free administration of cost-effective vaccines. A novel mucoadhesive bilayer film was developed to test the feasibility of this route of immunization in rabbits. METHODS: Bilayer films were developed using different ratios of Noveon and Eudragit S-100 as the mucoadhesive layer and a pharmaceutical wax as the impermeable backing layer. Optimal 3/8-inch films were post-loaded with 100 microg of plasmid DNA (CMV-beta-gal) or beta-galactosidase protein. The in vitro release rates and stability of the postloaded antigens were determined. The films were applied to the buccal pouch of rabbits on days 0. 7, and 14, and the humoral and splenocyte proliferative immune responses to beta-gal were determined through day 28 and compared to those responses after conventional subcutaneous injection of adjuvanted protein. RESULTS: The weight ratio of Noveon and Eudragit S-100 had a significant effect on adhesion time of bilayer films. Postloaded plasmid DNA and beta-gal remained stable after being released from bilayer films (release of -60-80% in 2 h for both). Buccal immunization using novel bilayer films (109 +/- 6-microm thickness) containing plasmid DNA led to comparable antigen-specific IgG titer to that of subcutaneous protein injection. All rabbits immunized with plasmid DNA via the buccal route but none by the subcutaneous route with protein antigen demonstrated splenocyte proliferative immune responses. CONCLUSION: The feasibility of buccal (genetic) immunization with these novel bilayer films was demonstrated.

Adhesives↗

PubMed2002

Buccal transmucosal delivery of calcitonin in rabbits using thin-film composites.

PURPOSE: Salmon Calcitonin (sCT) is used to treat hypercalcemia resulting from Paget's disease and osteoporosis. sCT is available either in a sterile injectable form or nasal spray. Alternative and more cost-effective dosage forms for the delivery of calcitonin are needed. We sought to deliver sCT transmucosally using a previously reported mucoadhesive bilayer thin-film composite (TFC) via the buccal route. METHODS: Forty micrograms of salmon calcitonin (200-IU) was loaded on preformed TFCs. In vitro release of sCT from TFCs was monitored in phosphate-buffered saline (10 mM, pH 7.4) at 37degrees C. Female New Zealand White rabbits (n = 6) were dosed with 40 microg of sCT either by injection via the ear vein or by applying sCT-loaded TFCs directly on the buccal pouch. Blood was collected at various times, and the plasma sCT and calcium concentrations were quantified. WinNonlin was used to determine the relevant pharmacokinetic parameters. RESULTS: In vitro, over 80% of sCT was released from the TFCs within 240 min. Super Case-II transport was indicated as the primary release mechanism. Rabbits injected intravenously had C(max), Cls, Vss, and AUC(0-inf) values of 75.1 +/- 6.5 ng/mL, 20.7 +/- 3.3 mL/min, 637 +/- 141 mL, and 1925 +/- 237 ng*min/mL, respectively. Rabbits dosed via the buccal route had C(max) Cls, and AUC(0-400 min values of 4.6 +/- 1.6 ng/mL, 22.0 +/- 5.9 mL/min, and 842.9 +/- 209.7 ng*min/mL, respectively. The relative bioavailability for rabbits treated with the TFCs was 43.8 +/- 10.9% with a CV of 24.9%. The reductions in plasma calcium levels after administration of sCT by both the intravenous and buccal route were comparable. CONCLUSIONS: The TFCs effectively delivered therapeutically efficacious amounts of sCT across the buccal mucosa in rabbits.

Administration, Buccal↗

PubMed2002

Intranasal administration of plasmid DNA-coated nanoparticles results in enhanced immune responses.

Intranasal immunization offers potential forthe elicitation of effective mucosal and systemic immune responses. In this study, a previously reported novel cationic nanoparticle engineered from a microemulsion precursor was further modified, optimized and applied intranasally to mice to explore its potential as a plasmid DNA (pDNA) vaccine delivery system. To this end, more uniform nanoparticles (around 100 nm) containing less cationic surfactant were developed. The pDNA-coated nanoparticles significantly enhanced the specific serum IgG and IgA titres to an expressed model antigen, beta-galactosidase, by 18-28 and 25-30 fold, respectively, when compared with naked pDNA alone. An enhanced splenocyte proliferative response was also observed after immunization with the pDNA-coated nanoparticles. It was concluded that these plasmid DNA-coated nanoparticles may have potential for immunization via the nasal route.

Administration, Intranasal↗

PubMedDate not supplied

Plasmid DNA-entrapped nanoparticles engineered from microemulsion precursors: in vitro and in vivo evaluation.

Nonviral gene therapy has been a rapidly growing field. However, delivery systems that can provide protection for pDNA and potential targeting are still desired. A novel pDNA-nanoparticle delivery system was developed by entrapping hydrophobized pDNA inside nanoparticles engineered from oil-in-water (O/W) microemulsion precursors. Plasmid DNA was hydrophobized by complexing with cationic surfactants DOTAP and DDAB. Warm O/W microemulsions were prepared at 50-55 degrees C with emulsifying wax, Brij 78, Tween 20, and Tween 80. Nanoparticles were engineered by simply cooling the O/W microemulsions containing the hydrophobized pDNA in the oil phase to room temperature while stirring. The nanoparticles were characterized by particle sizing, zeta-potential, and TEM. Nanoparticles were challenged with serum nucleases to assess pDNA stability. In addition, the nanoparticles were coincubated with simulated biological media to assess their stability. In vitro hepatocyte transfection studies were completed with uncoated nanoparticles or nanoparticles coated with pullulan, a hepatocyte targeting ligand. In vivo biodistribution of the nanoparticles containing I-125 labeled pDNA was monitored 30 min after tail-vein injection to Balb/C mice. Depending on the hydrophobizing lipid agent employed, uniform pDNA-entrapped nanoparticles (100-160 nm in diameter) were engineered within minutes from warm O/W microemulsion precursors. The nanoparticles were negatively charged (-6 to -15 mV) and spherical. An anionic exchange column was used to separate unentrapped pDNA from nanoparticles. Gel permeation chromatography of pDNA-entrapped and serum-digested nanoparticles showed that the incorporation efficiency was approximately 30%. Free 'naked' pDNA was completely digested by serum nucleases while the entrapped pDNA remained intact. Moreover, in vitro transfection studies in Hep G2 cells showed that pullulan-coated nanoparticles resulted in enhanced luciferase expression, compared to both pDNA alone and uncoated nanoparticles. Preincubation of the cells with free pullulan inhibited the transfection. Finally, 30 min after tail vein injection to mice, only 16% of the 'naked' pDNA remained in the circulating blood compared to over 40% of the entrapped pDNA. Due to the apparent stability of these pDNA-entrapped nanoparticles in the blood, they may have potential for systemic gene therapy applications requiring cell and/or tissue-specific delivery.

Animals↗