Transgenic tobacco plants stably expressing recombinant FaeG, which is the major subunit and adhesin of K88ad fimbriae, were obtained. Analysis of sera from immunized mice indicates that in mice, the immunogenicity induced by plant-derived FaeG protein is comparable to that generated with traditional approaches.Enterotoxigenic Escherichia coli (ETEC) strains are commonly associated with neonatal diarrhea in piglets (6,11,12,13,19). Among the different ETEC strains, those expressing K88 fimbrial antigen are the most prevalent type (1, 18). Fimbriae (or pili) are the primary pathogenicity factors of this bacterium and are responsible for its adhesion to enterocyte receptors. Analysis of the genetic organization of the K88 gene cluster has revealed that at least eight structural genes are involved in biosynthesis. FaeG (27.6 kDa) is the so-called major fimbrial subunit protein that also carries the adhesive properties of the K88 fimbriae (4,17,24,26).Vaccines containing purified K88 fimbriae, formalin-inactivated ETEC, or engineered E. coli expressing K88 fimbriae have been used to vaccinate pregnant sows, and passive transfer of lacteal immunity from the vaccinated dams can protect piglets from ETEC infection (5,10,15,21,25,27). Although proven effective for the prevention of disease, limiting the widespread use of these vaccines are the fact that bacteria might not be inactivated fully and the high cost of producing and preserving these vaccines.Recently, the use of plants as bioreactors has become of special interest, as they allow production of recombinant proteins in large quantities at relatively low cost (14,22,23,30). For the development of FaeG-based vaccine against K88 ETEC strains in plants, we constructed pBI8801, a plant binary expression vector containing the K88ad fimbrial major antigen gene (faeG). First, p8801 (31), a parental plasmid which contains faeG without a signal peptide coding region, was digested with BamHI and SacI and the 789-bp faeG fragment was cloned into the digested plant expression vector pBI 121 (Clontech, Palo Alto, Calif.). This led to the creation of pBI8801, a binary vector with a cauliflower mosaic virus (CaMV 35s) promoter and a nopaline synthase terminator. Triparental matings were then performed as described by Ditta et al. (7) to transfer plasmid pBI8801 into Agrobacterium tumefaciens LBA4404.Thereafter, transgenic tobacco (Nicotiana tabacum) was obtained by a modified leaf-disk cocultivation method using A. tumefaciens harboring pBI8801 (16). PCR analysis was carried out to show the presence of an amplified product of the expected size (789 bp) in the genomic DNAs (8) of tested kanamycin-resistant plants (data not shown). Reverse transcriptase PCR (RT-PCR) was used according to the protocol of a PowerScript RT kit (Clontech) to test whether faeG was transcribed in the transgenic plants; a fragment with expected size was observed for all the tested transformants, whereas no product was observed for nontransgenic plants (Fig. 1A).The transgenic plants were further tested ...

To increase the cellular uptake and drug loading of cellulose nanocrystal (CNC)-based nanomedicines, folate/cis-aconityl-doxorubicin@polyethylenimine@CNC (FA/CAD@PEI@CNC) nanomedicines were built up by the building blocks of folate (FA), cis-aconityl-doxorubicin (CAD), polyethylenimine (PEI), and CNCs via the robust layer-by-layer (LbL) assembly technique. The drug loading content (DLC) of FA/CAD@PEI@CNC hybrids was 11.3 wt %, which was almost 20-fold higher than that of the CNC-based nano-prodrug we reported previously. FA/CAD@PEI@CNC nanomedicines showed lysosomal pH-controlled drug release profiles over 24 h. In detail, the cumulative drug release was over 95% at pH 5.5, while the cumulative drug release was only 17% at pH 7.4. In vitro, FA/CAD@PEI@CNC hybrid nanomedicines had a higher (9.7-fold) mean fluorescent intensity (MFI) than that of DOX·HCl, with enhanced cytotoxicity and decreased IC50 against MCF-7. Thus, FA/CAD@PEI@CNC hybrid nanomedicines displayed efficient targetability and enhanced cellular uptake. In addition, FA/CAD@PEI@CNC nanomedicine could deliver more DOX to the nucleus than the control group, due to the β-carboxylic acid catalyzed breakage of the pH-labile cis-aconityl amide linkages in CAD. These results indicated that FA/CAD@PEI@CNC nanomedicines achieved lysosomal pH-controlled drug release into the nucleus and showed great potential to be high-performance nanomedicines to improve the delivery efficiency and therapy efficacy. This study for CNC-based nanomedicines provided important insights into the bioapplication of CNCs modified by LbL assembly.

A biodegradable brush-type copolymer PHF-g-(PCL-PEG) based on a cleavable polyacetal backbone and biodegradable side chain modified with polyethylene glycol (PEG) was synthesized in this paper. This particular structure was directional to facilitate the formation of spherical or rod-shaped micelles. Flow cytometry showed that rod-shaped micelles displayed enhanced cellular uptake compared to spherical micelles. Rod-shaped micelles were selected to investigate their drug delivery abilities in detail. In vitro experiments verified the pH-triggered drug release of DOX-loaded micelles, and the release rate of doxorubicin (DOX) was 77% at pH 5.0 and 26% at pH 7.4. In drug-release kinetic analysis, a double-exponential model achieved the best fit. The copolymer appeared to be almost nontoxic, while the DOX-loaded micelles showed equivalent cytotoxicity compared to DOX at high concentration. The endocytosis of DOX-loaded micelles was two times that of DOX. Our findings suggest that the pH-sensitive brush type copolymer could be a possible carrier in drug delivery.

SYNOPSISPoly(N-isopropylacrylamide) (PIPA) was synthesized by radical polymerization with 2,2'-azobisisobutyronitrile (AIBN) as an initiator and 3-mercaptopropionic acid (MPA) as a chain-transfer reagent in methanol (MeOH) at 70°C for 7 h. The resultant PIPA was grafted to polyallylamine hydrochloride (PAlAm -HCl) by amide formation under the influence of water-soluble carbodiimide l-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC). The graft polymer was made into microspheres (MS) by chemical crosslinking. The pH-responsive drug release of the graft polymer microspheres was examined by releasing phenobarbital natrium (PN), which was carried on the microspheres by physical adsorption. A dynamic dialysis technique was used in the drug-release experiment and the drug-release-rate constants reflecting the drug release characteristic of polymer microspheres were obtained by constituting a mathematical model. The results indicated that the homopolymer PAlAm microspheres and the copolymer PAIAm-g-PIPA microspheres are both pH responsive to release P N and that in the neutral pH condition the release rate is the slowest. 0 1995 John Wiley & Sons, Inc. INTRODUCTIONThe external signal-responsive drug-delivery system has been paid more and more attention. The obvious advantage of this system is the "on-off" switching control release of a drug from a drug carrier by surrounding signals such as heat, chemical compounds, electric field, and pH. Many polymers such as polyacrylamide,' poly(N-acryloylpyrrolidine),2 poly(N-alkyl-substituted acrylamides)? and some liposomes4 can be used to achieve this "onoff'' drug-control release as a drug-carrier martrix. Among these polymers, poly(N-isopropylacrylamide)5 (PIPA) has demonstrated noticeable thermosensitivity in terms of water swelling. Okahata et a1.6 even used a large nylon capsule membrane with a surface-grafted poly(N-isopropylacrylamide) to regulate reversily the permeation of NaCl and dyes by ambient temperature change. Recently, Schild7 reported a detailed review of the specificity, synthesis, and application of PIPA. Changes in the swelling states of PIPA gels can influence the diffusion of solutes from within the gels to the outside aqueous media. The changes were mainly thermosensitive. However, the review reported also a novel e x t e n~i o n~'~ of the PIPA system in which acrylic acid as a comonomer was introduced consequently, the copolymer possessed not only a thermal response but also pH sensitivity. The lower critical solution temperature (LCST) shifts to higher temperature at higher pH due to repulsion between the ionized groups. Yan" also utilized PIPA to obtain controllable catalytic activity of the enzyme immobilized by the medium temperature. According to Yan's work, the phase transition of the graft copolymer polyallylamine hydrochloride (PAlAm) -g-PIPA was influenced not only by the temperature but also by the pH of the dissolution medium. Extensively, we carried out drug-release experiments with PAIAmg-PIPA as a drug carrier at various pH's....

Poly(lactide-co-glycolide) (PLGA 75 : 25), IV 0.94 dL/g was chosen as the matrix of the microparticles. Bovine serum albumin (BSA) (Fraction V) as the model drug was incorporated in the microparticles by a W/O/W emulsification and solvent evaporation technique. The effect of the various preparation parameters on particle morphology, drug loading efficiency, and drug release profiles of the resultant microparticles were examined. Particle size varied from 5 to 60 m. The final morphology of the microparticles varied dramatically with preparation variables such as equipment used to produce the primary emulsion (W1/O) and the water-to-oil ratio (W1/O) in the primary emulsion. In general, the viscosity of the primary emulsion had a significant effect on the porosity of particles produced. The release of BSA showed a strong relationship with the preparation parameters of microparticles, partly due to the morphological effects. For example, microparticles made from the vortex mixer that was used to disperse inner aqueous phase (W1) to oil phase (O) showed a lower burst effect than that made from the homogenizer because of its better surface morphology. W1/O ratio, speed of dispersing the primary emulsion into W2, PLGA concentration, and different matrix materials also affected the drug release profiles. In all the samples studied here, only diffusion-controlled release was observed.