Production and characterization of liquid-core capsules made from cross-linked acrylamide copolymers for biotechnological applications

A novel chemistry has been developed for the production of capsules composed of a hydrophobic liquid core surrounded by a cross-linked polyacrylamide/alginate membrane. These liquid-core capsules may be used in capsular perstraction for the removal of inhibitory products from bioprocesses and bioconversions. They have the advantage of having a high surface area to promote rapid mass transfer, while separation of the organic core phase from the aqueous environment by the capsule membrane prevents the formation of stable emulsions and potential problems associated with toxicity of the organic phase for microbial cells or enzymes. Monodisperse spherical liquid-core capsules of between 800 microm and 1.6 mm diameter, with high mechanical resistance, have been prepared by co-extrusion, using the jet break-up technique. Capsules produced from a solution of MBA/total monomer (5%) were found to be more elastic and have a higher burst force when exposed to chelating agents such as phosphate or citrate. The mechanical resistance was unaffected by buffer solutions in the pH range 4-9 and after sterilization at 121 degrees C for 20 min. Capsules having membranes composed of a copolymer of acrylamide and N-hydroxymethylacrylamide exhibited even higher mechanical stability toward chelating agents.     

A novel reactive perstraction system based on liquid-core microcapsules applied to lipase-catalyzed biotransformations

A novel reactive perstraction system has been developed based on liquid-core capsules, involving an enzyme-catalyzed reaction coupled with simultaneous in situ product recovery. Lipase-catalyzed reactions, hydrolysis of triprionin and nitrophenyl laurate, were selected to test the system and demonstrate the feasibility of immobilization of enzymes to the membranes of liquid-core capsules and the ability to extract hydrophobic products of the reaction within the capsule core. The lipase from Candida rugosa was immobilized to the microcapsules by adsorption and by covalent binding through activation with glutaraldehyde. In both cases improved temperature and operational stability were achieved. Both types of immobilization resulted in a basic shift of the pH optimum for activity, from 7.5 to 9.0. The presence of an organic phase within the capsule core allowed direct product separation and lead to a decrease in product inhibition of the lipase-catalyzed reaction.     

Encapsulation of mammalian cell with chitosan-CMC capsule

Viable hybridoma cells were encapsulated. The capsules were formed in one step by placing a drop of cell suspension mixed with negatively charged carboxymethylcellulose (CMC) into a positively charged chitosan solution through the interpolymeric ionic interaction between two oppositely charged polymers. These capsules were found to have a mean diameter of about 1. 5 mm and wall thickness of 3 mum. The cells grew in the capsules using supplemented DMEM/F12 (four kinds of growth factor). The maximum cell density in encapsulating cell culture reached 1 x 10(7) cells/ml, 10 times higher than that obtained in the free cell culture. The maximum monoclonal antibody concentration in the free cell culture was 15mug/mL, but that in the capsule was 45mug/mL The antibody produced by the cell was concentrated about four times higher inside than outside of the capsules.     

Characterisation of small molecules diffusion in hydrogel-membrane liquid-core capsules

The overall diffusion coefficients for several low molecular weight solutes, such as glucose, fructose, sucrose, lactose, and vitamin B(12) have been determined in Ca-alginate membrane liquid-core capsules using the unsteady-state method following the release of solutes from the capsules to a well-stirred solution of limited volume. The diffusion coefficients obtained for saccharides were 5-20% lower than the corresponding diffusivity in water while for vitamin B(12) about 50% that of water. The diffusion coefficients of the investigated capsules were not influenced by the change in alginate concentration in the capsule membrane from 0.5 to 1.0%. Lower diffusivities and higher deviations from the diffusivity in water were obtained for higher molecular weight solutes.     

Microencapsulation of recombinant Saccharomyces cerevisiae cells with invertase activity in liquid-core alginate capsules

As a means of integrating cell growth and immobilization, recombinant Saccharomyces cerevisiae cells with invertase activity were immobilized in liquid-core alginate capsules and cultured to a high density. S. cerevisiae cells of SEY 2102 (MAT alpha ura3-52 leu2-3, 112 his4-519) harboring plasmid pRB58 with the SUC2 gene coding for invertase were grown to 83 g/L of liquid-core volume inside the capsule on a dry weight basis. The cloned invertase was expressed well in the immobilized cells with slightly higher activity than the free cells in a batch culture. Invertase in the immobilized cells showed slightly more improved thermal stability than in the free cells. Storage in a Na-acetate buffer at 4 degrees C and 10 degrees C for 1 month resulted in 7% and 8% loss in activity, respectively. The sucrose hydrolysis reaction was stably maintained for 25 repeated batches for 7 days at 30 degrees C. Continuous hydrolysis of 0.3 M sucrose was carried out in a packed bed reactor with a conversion of more than 90% at a maximum productivity of 55.5 g glucose/L per hour for 7 days. In a continuous stirred tank reactor, the maximum productivity of 80.8 g glucose/L per hour was achieved at a conversion of 59.1% using 1.0 M sucrose solution, and 0.5 M sucrose solution was hydrolyzed for 1 week with a 95% conversion at a productivity of 48.8 g/L per hour. (c) 1996 John Wiley & Sons, Inc.     

Calculations on O2 transfer in capsules with animal cells for the determination of maximum capsule size without O2 limitation

The size of the capsules without O2 limitation and maximum allowable capsule size for different cell densities were calculated by the observable modulus (modified Thiele modulus). When hybridoma (ATCC HB-8852) at 2.5 3 × 105cells/ ml was encapsulated and grown, the cell density reached to 2.6 × 107cells/ml in the 7th day of incubation. The average diameter of the capsule was 2.1mm. The maximum cell density obtained from experiment agreed with the calculated cell density for the given size of the capsules. © Rapid Science Ltd. 1998     

Encapsulation of <Emphasis Type="Italic">Pannonibacter phragmitetus</Emphasis> LSSE-09 in alginate–carboxymethyl cellulose capsules for reduction of hexavalent chromium under alkaline conditions

Cr(VI) was efficiently reduced to Cr(III) by Pannonibacter phragmitetus LSSE-09 encapsulated in liquid-core alginate–carboxymethyl cellulose capsules under alkaline conditions. Taking into account the physical properties of the capsules, the activity of encapsulated cells, and total Cr(III) concentration in the supernatant, optimal conditions (0.5% w/v sodium alginate; 2% w/v sodium carboxymethyl cellulose; 0.1 M CaCl₂; 30-min gelation time) for LSSE-09 encapsulation were determined. At optimal conditions, a relatively high reduction rate of 4.20 mg g_{(dry weight)}⁻¹  min⁻¹ was obtained. Total Cr(III) concentration in the supernatant was significantly decreased after reduction, because 63.7% of the formed soluble organo-Cr(III) compounds compared with those of free cells were captured by the relatively smaller porous structure of alginate capsules. The optimal pH value (9.0) for Cr(VI) reduction was not changed after encapsulation. In addition, encapsulated LSSE-09 showed no appreciable loss in activity after eight repeated cycles at 37°C, and 85.7% of its initial activity remained after 35-day storage at 4°C. The results suggest that encapsulated LSSE-09 in alginate–carboxymethyl cellulose capsules has potential biotechnological applications for the detoxification of Cr(VI)-contaminated wastewater.     

Regulation of antibody secretion by hybridoma cells. I. Suppression of antibody secretion by coculture of hybridoma cells with idiotype-induced suppressor cells

In this study, we have demonstrated that antibody secretion by hybridoma cell lines can be down-regulated by idiotype-specific immune spleen cells or by nylon wool nonadherent spleen cells. This suppression of antibody secretion can be abolished by treating the idiotype-specific immune spleen cells with anti-Thy 1.2 plus complement. The hybridoma we used for most of our experiments secretes IgM specific for the cross-reacting haptens 2,4,6-trinitrophenyl (TNP) and 2,4-dinitrophenyl (DNP). Suppression was achieved by direct coculture of hybridoma cells with immune cells from animals which were injected with affinity-purified hybridoma antibody-coupled syngeneic spleen cells. The suppressed and control cultures contained similar numbers of viable hybridoma cells, suggesting that a simple cytotoxic effect is not responsible. Idiotype specificity was established in experiments showing that two idiotype immune animals immunized with antibody from two different IgM anti-TNP hybridomas could suppress the hybridoma to which they were immunized but could not affect the other hybridoma. Immune spleen cells required 3-4 days of coculture with hybridoma cells before maximum suppression was achieved. The kinetics of the response suggest that the final effector suppressor cell is generated during the coculture period and that a second signal, perhaps a product of the hybridoma cells, may be required.     

抗麻痹性贝毒素GTX2,3单克隆抗体的制备及特性分析

制备抗麻痹性贝毒GTX2,3单克隆抗体。利用醛化法将GTX2,3与载体牛血清白蛋白(BSA)偶联,制备完全抗原。免疫小鼠,取小鼠脾细胞与Sp2/0细胞融合。GTX2,3与钥孔血蓝蛋白(KLH)偶联作为检测抗原,用间接ELISA法筛选阳性克隆株。将筛选的阳性细胞株制备腹水。获得三株稳定分泌抗GTX2,3单克隆抗体的杂交瘤细胞株F4、F10、G9。间接ELISA法检测F10细胞株腹水抗体效价为1.4×10^-5。半抗原GTX2,3与载体蛋白偶联后,作为免疫原,可制备高滴度的抗GTX2,3抗血清和单克隆抗体。该抗体对于藻毒素具有高特异性和高亲和力,可用于污染海产品的麻痹性贝毒的检测。     

High‐Efficiency Polymer Homo‐Tandem Solar Cells with Carbon Quantum‐Dot‐Doped Tunnel Junction Intermediate Layer

The tunnel junction (TJ) intermediate connection layer (ICL), which is the most critical component for high‐efficient tandem solar cell, generally consists of hole conducting layer and polyethyleneimine (PEI) polyelectrolyte. However, because of the nonconducting feature of pristine PEI, photocurrent is open‐restricted in ICL even with a little thick PEI layer. Here, high‐efficiency homo‐tandem solar cells are demonstrated with enhanced efficiency by introducing carbon quantum dot (CQD)‐doped PEI on TJ–ICL. The CQD‐doped PEI provides substantial dynamic advantages in the operation of both single‐junction solar cells and homo‐tandem solar cells. The inclusion of CQDs in the PEI layer leads to improved electron extraction property in single‐junction solar cells and better series connection in tandem solar cells. The highest efficient solar cell with CQD‐doped PEI layer in between indium tin oxide (ITO) and photoactive layer exhibits a maximum power conversion efficiency (PCE) of 9.49%, which represents a value nearly 10% higher than those of solar cells with pristine PEI layer. In the case of tandem solar cells, the highest performing tandem solar cell fabricated with C‐dot‐doped PEI layer in ICL yields a PCE of 12.13%; this value represents an ≈15% increase in the efficiency compared with tandem solar cells with a pristine PEI layer.     

Encapsulated animal cell culture for the production of monoclonal antibody (MAb)

Biopolymer membrane was prepared using two oppositely charged natural biopolymer. The biopolymer membrane was used for the encapsulation of two hybridoma cell (ATCC CRL-1606, ATCC BH-8852) to produce monoclonal antibodies. In order to reduce the down stream steps, the pore size of the membrane was controlled to retain the monoclonal antibodies in the capsules based on the diffusion experiments with standard proteins. T-flask culture showed cell densities of 8×10⁷ cells/mL and 3×10⁷ cells/mL, and MAb concentrations of 506 μg/mL and 109 μg/mL for encapsulated ATCC CRL-1606 and HB-8852, respectively. Two liter perfusion culture with encapsulated ATCC HB-8852 was performed to enhance the MAb production. The MAb production of the encapsulated hybridoma increased considerably comparing to the culture using silicone tubing for oxygen transfer.     

新型阳离子基因传递载体PEI-PBLG的细胞转染研究

对新型阳离子聚合物PEI(10kD)-PBLG进行研究,重点考察其基因转染效率与细胞毒性,探讨其作为基因载体的可能性。通过粒径分析及扫描电镜(SEM)观察PEI(10kD)-PBLG与质粒pEGFP自组装形成的颗粒形态及粒径,预测其进入细胞的可能性。使用MTT比色法分析PEI(10kD)-PBLG、PEI(25kD)-PBLG、PEI(10kD)和PEI(25kD)的细胞毒性差异。选用表达增强型绿色荧光蛋白的质粒pEGFP作为报告基因模型,将其与PEI(10kD)-PBLG自组装后,分别转染真核细胞株Hela、COS-7、Vero-E6和ECV304,应用流式细胞术检测细胞转染效率,并比较了血清、缓冲液、细胞谱等多种因素对基因转染效率的影响。PEI(10kD)-PBLG可包裹质粒形成粒径100~120nm的纳米复合物,适合介导质粒进入细胞。该纳米粒复合物对转染缓冲液的敏感度较低,并能够在10%血清存在的条件下,转染全部实验用细胞株,尤其对Hela的转染效率最高,其次是COS-7、Vero-E6和ECV304;其中PEI-PBLG(10kD)/pEGFP复合物转染Hela细胞的比率为45.02%,高于PEI(10kD)/pEGFP的29.16%;PEI(10kD)-PBLG的细胞毒性作用显著低于PEI(25kD)、PEI(10kD)和PEI(25kD)-PBLG。新型阳离子多聚物PEI(10kD)-PBLG在提高PEI介导的基因转染效率的同时降低了其细胞毒性,提高了生物相容性,有望成为基因转移的有效载体。     

Whole cell enzyme microencapsulation of Escherichia coli with oxygen-dependent inducible nar promoter

As a means of integrating cell growth and immobilization, Escherichia coli with the cloned nar promoter on the pBR322 plasmid, which is maximally induced under anaerobic conditions in the presence of nitrate, was immobilized in liquid-core alginate capsules and cultured to a high cell density. The total -galactosidase activity obtained by immobilized cells was about 6 fold greater than that obtained by free cells. Using the immobilized -galactosidase in the whole cells, the substrate lactose was hydrolyzed to glucose and galactose stably with a conversion efficiency of more than 80% for 15 repeated batches at 30°C for 5 days.     

Low molecular weight polyethylenimine for efficient transfection of human hematopoietic and umbilical cord blood-derived CD34+ cells

With the emerging role of hematopoietic stem cells as potential gene and cell therapy vehicles, there is an increasing need for safe and effective nonviral gene delivery systems. Here, we report that gene transfer and transfection efficiency in human hematopoietic and cord blood CD34+ cells can be enhanced by the use of low molecular weight polyethylenimine (PEI). PEIs of various molecular weights (800-750,000) were tested, and our results showed that the uptake of plasmid DNA by hematopoietic TF-1 cells depended on the molecular weights and the N/P ratios. Treatment with PEI 2K (m.w. 2000) at an N/P ratio of 80/1 was most effective, increasing the uptake of plasmid DNA in TF-1 cells by 23-fold relative to Lipofectamine 2000. PEI 2K-enhanced transfection was similarly observed in hematopoietic K562, murine Sca-1+, and human cord blood CD34+ cells. Notably, in human CD34+ cells, a model gene transferred with PEI 2K showed 21,043- and 513-fold higher mRNA expression levels relative to the same construct transfected without PEI or with PEI 25 K, respectively. Moreover, PEI 2K-treated TF-1 and human CD34+ cells retained good viability. Collectively, these results indicate that PEI 2K at the optimal N/P ratio might be used to safely enhance gene delivery and transfection of hematopoietic and human CD34+ stem cells.     

Poly(ethyleneimine)-mediated large-scale transient gene expression: influence of molecular weight, polydispersity and N-propionyl groups

Three synthesis lots of linear poly(ethyleneimine) (PEI) are compared to a fully hydrolyzed linear PEI (commercially available as PEI "Max") regarding structure, polyplex formation with plasmid DNA, and transfection of suspension-adapted HEK-293E cells. PEI "Max" binds DNA more efficiently than the other PEIs, but it is the least effective in terms of transient recombinant protein yield. One PEI lot is fractionated by means of SEC. The fractions of high-M(n) PEI are the most efficient for complex formation and transfection. Nevertheless, the highest transient recombinant protein yields are achieved with unfractionated PEI. The results demonstrate that the polydispersity and charge density of linear PEI are important parameters for gene delivery to suspension-adapted HEK-293E cells.     

Novel reactive perstraction system applied to the hydrolysis of penicillin G

The activity of penicillin acylase has been studied in aqueous and organic solvents, as free enzyme as well as immobilized within the membrane of liquid-core capsules. The activity of the enzyme is inhibited by the accumulation of the products of the hydrolysis reaction, namely phenyl acetic acid (PAA). In order to overcome this inhibition a range of organic solvents were tested for use in in situ product recovery. Of these solvents dibutyl sebacate (DBS) was chosen due to the rapid extraction rate, the high logP and to facilitate capsule production. The extraction efficiency at pH 3.5 for PAA was >80% for phase ratios of >50% free solvent with partition coefficients of 8 and 0.7 for PAA and penicillin G (PenG), respectively, thereby showing that PAA could be selectively extracted at pH 3.5 and 25 degrees C. Liquid-core capsules containing DBS were shown to efficiently remove PAA selectively and the PAA could be effectively back-extracted and the capsules re-used in a three-stage process resulting in high product separation. Immobilization of penicillin acylase onto the capsule membranes resulted in increased operational stability of the enzyme and a very high enzyme activity. Over 53.3% of the PAA formed could be recovered in the capsule core with a concentration over sevenfold higher than in the aqueous phase. Higher extraction efficiencies could be obtained by varying the substrate concentration and number of capsules. The enzyme immobilized on capsules could be stored for over 4 months at pH 8 and 4 degrees C with no loss of activity. Over 80% of the initial activity could be recovered over five repeated batch cycles of the bioconversion process. The importance of capsular perstraction and reactive capsular perstraction has been clearly demonstrated.     

Asymmetric synthesis of duloxetine intermediate (S)-(-)-3-N-methylamino-1-(2-thienyl)-1-propanol using immobilized Saccharomyces cerevisiae in liquid-core sodium alginate/chitosan/sodium alginate microcapsules

Duloxetine intermediate (S)-(-)-3-N-methylamino-1-(2-thienyl)-1-propanol was synthesized using ACA liquid-core immobilized Saccharomyces cerevisiae CGMCC No. 2230. The optimum culture time for ACA liquid-core immobilized cells was found to be 28 h. The optimum ACA liquid-core capsule formation conditions were found to be 90 % chitosan deacetylation, 30,000–50,000 chitosan molecular weight, 5.0 g/L chitosan, and pH 6.0 citrate buffer solution. The highest activity was found when reduction conditions were pH 6.0, 30 °C and 180 rpm. The ACA-immobilized cells can be reused nine times and only 40 % of the activity is retained after nine cycles. Product inhibition of reduction was observed in batch reduction. Continuous reduction in the membrane reactor was found to remove the product inhibition on reduction and improve production capacity. Conversion reached 100 % and enantiometric excess of (S)-(-)-3-N-methylamino-1-(2-thienyl)-1-propanol exceeded 99.0 % in continuous reduction of 5 g/L 3-N-methylamino-1-(2-thienyl)-1-propanone in the membrane reactor.     

Lactosylated polyethylenimine for gene transfer into airway epithelial cells: role of the sugar moiety in cell delivery and intracellular trafficking of the complexes

BACKGROUND: As we have previously shown that lactosylated polyethylenimine (PEI) is the most efficient glycosylated PEI for gene transfer into human airway epithelial cells in primary culture, we have studied here the role of the lactose residue in the enhancement of gene transfer efficiency observed with lactosylated PEI as compared with unsubstituted PEI in immortalized (Sigma CFTE29o- cells) and primary human airway epithelial cells. METHODS AND RESULTS: After three transfections of 1 h performed daily, 60% of Sigma CFTE29o- cells were transfected with lactosylated PEI, whereas 25% of cells were transfected with unsubstituted PEI (p < 0.05). Cell viability was 1.8-fold greater with lactosylated PEI as compared with unsubstituted PEI (p < 0.05). As assessed by flow cytometry, the cellular uptake of lactosylated complexes was greater than that of complexes made with unsubstituted PEI (p < 0.05) and involved mostly a receptor-mediated endocytosis. The study of the intracellular trafficking in airway epithelial cells of complexes showed an endosomal and lysosomal accumulation of lactosylated complexes. In the presence of a proton pump inhibitor, the level of lactosylated and unsubstituted PEI-mediated gene expression was reduced more than 20-fold, whereas the cell viability increased to almost 100%. For both complexes, a nuclear localization was observed for less than 5% of intracellular complexes. CONCLUSIONS: Our results show that the greater gene transfer efficiency observed for lactosylated complexes may be attributed to a higher amount of lactosylated complexes incorporated by airway epithelial cells and a lower cytotoxicity that might be related to reduced endosomolytic properties. However, the lactose residues substituting the PEI did not promote the entry of the plasmid into the nucleus.     

Intraperitoneal administration of poly(I:C) with polyethylenimine leads to significant antitumor immunity against murine ovarian tumors

Ovarian cancer is currently the most lethal gynecologic cancer in the United States. There is an urgent need for the development of innovative therapies against ovarian cancer, such as immunotherapy. The toll-like receptor 3 ligand, polyriboinosinic:polyribocytidylic acid (poly(I:C), has emerged as a promising adjuvant for activating the host immune responses for the control of tumors. We reasoned that a strategy to enhance the intracellular uptake of poly(I:C) will likely improve the poly(I:C) adjuvant effect. Since polyethylenimine (PEI) has been shown to increase the transfection efficiency of nucleic acids, we characterized the antitumor effects in mouse ovarian surface epithelial cells (MOSEC) tumor-bearing mice treated intraperitoneally with poly(I:C) and PEI. We observed that tumor-bearing mice treated with poly(I:C) and PEI generated significantly better therapeutic antitumor effects against MOSEC tumors compared with treatment with poly(I:C) alone. Furthermore, we found that NK cells play a significant role in the antitumor effects generated by treatment with poly(I:C) in combination with PEI. Intraperitoneal administration of poly(I:C) with PEI led to the uptake of poly(I:C) mainly by CD11b+ macrophages, resulting in the high expression of MHC class II and IL-12 (M1 phenotype). In addition, adoptive transfer of CD11b+ macrophages from mice treated with poly(I:C) and PEI was found to lead to increased number of activated NK cells in the recipient mice. Taken together, our data indicate that PEI can potentially be used to improve the uptake of poly(I:C) by CD11b+ macrophages, leading to the activation of NK cells and the control of murine ovarian tumors.     

Targeted double domain nanoplex based on galactosylated polyethylenimine enhanced the delivery of IL-12 plasmid

The objective of the present investigation was to design a targeted polyethylenimine (PEI)-based polyplex by conjugating lactose bearing galactose groups on low molecular weight PEI (LMW PEI) grafted to a high molecular weight PEI (HMW PEI) via a succinic acid linker in order to restore the amine content of the whole conjugate used for ligand conjugation. The PEI conjugate was synthesized and characterized in terms of buffering capacity, particle size, zeta potential, plasmid condensation ability, and protection of DNA against degrading enzymes. Also, the transfection efficiency and cytotoxicity were evaluated in the cell line over-expressing asialoglycoprotein receptors (ASGPRs) and compared with the cells lacking the receptors. The results demonstrated the ability of PEI conjugate in condensation of plasmid DNA and protection against enzyme degradation. The PEI conjugate formed nanoparticles of around 75 nm with higher buffering capacity compared with unmodified PEI. The polyplexes prepared by the modified PEI could increase the level of transgene up to four folds in the cells over-expressing the receptor. The results demonstrated the separation of targeting and delivery domains could be considered as a strategy to restore the amine content of the PEI molecule utilized for targeting ligand conjugation.