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1.
Appl Microbiol Biotechnol ; 102(11): 4829-4841, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29675801

RESUMO

This article comparatively reports the workability of Escherichia coli BL21(DE3) and Pseudomonas putida KT2440 cell factories for the expression of three model autodisplayed cellulases (i.e., endoglucanase, BsCel5A; exoglucanase, CelK; ß-glucosidase, BglA). The differentiation of the recombinant cells was restricted to their cell growth and enzyme expression/activity attributes. Comparatively, the recombinant E. coli showed higher cell growth rates but lower enzyme activities than the recombinant P. putida. However, the endo-, exoglucanase, and ß-glucosidase on the surfaces of both cell factories showed activity over a broad range of pH (4-10) and temperature (30-100 °C). The pH and temperature optima were pH 6, 60 °C (BsCel5A); pH 6, 60-70 °C (CelK); and pH 6, 50 °C (BglA). Overall, the P. putida cell factory with autodisplayed enzymes demonstrated higher bioactivity and remarkable biochemical characteristics and thus was chosen for the saccharification of filter paper. A volumetric blend of the three cellulases with P. putida as the host yielded a ratio of 1:1:1.5 of endoglucanase, exoglucanase, and ß-glucosidase, respectively, as the optimum blend composition for filter paper degradation. At an optical density (578 nm) of 50, the blend generated a maximum sugar yield of about 0.7 mg/ml (~ 0.08 U/g) from Whatman filter paper (Ø 6 mm, ~ 2.5 mg) within 24 h.


Assuntos
Celulases/genética , Escherichia coli/genética , Pseudomonas putida/genética , Celulases/biossíntese , Microbiologia Industrial , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética
2.
Crit Rev Biotechnol ; 37(2): 251-261, 2017 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26863480

RESUMO

Endotoxin is a type of pyrogen that can be found in Gram-negative bacteria. Endotoxin can form a stable interaction with other biomolecules thus making its removal difficult especially during the production of biopharmaceutical drugs. The prevention of endotoxins from contaminating biopharmaceutical products is paramount as endotoxin contamination, even in small quantities, can result in fever, inflammation, sepsis, tissue damage and even lead to death. Highly sensitive and accurate detection of endotoxins are keys in the development of biopharmaceutical products derived from Gram-negative bacteria. It will facilitate the study of the intermolecular interaction of an endotoxin with other biomolecules, hence the selection of appropriate endotoxin removal strategies. Currently, most researchers rely on the conventional LAL-based endotoxin detection method. However, new methods have been and are being developed to overcome the problems associated with the LAL-based method. This review paper highlights the current research trends in endotoxin detection from conventional methods to newly developed biosensors. Additionally, it also provides an overview of the use of electron microscopy, dynamic light scattering (DLS), fluorescence resonance energy transfer (FRET) and docking programs in the endotoxin-protein analysis.


Assuntos
Endotoxinas/análise , Animais , Técnicas Biossensoriais , Endotoxinas/metabolismo , Humanos , Proteínas/metabolismo
3.
Crit Rev Biotechnol ; 36(6): 1010-1022, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26381238

RESUMO

The genome of virulent strains may possess the ability to mutate by means of antigenic shift and/or antigenic drift as well as being resistant to antibiotics with time. The outbreak and spread of these virulent diseases including avian influenza (H1N1), severe acute respiratory syndrome (SARS-Corona virus), cholera (Vibrio cholera), tuberculosis (Mycobacterium tuberculosis), Ebola hemorrhagic fever (Ebola Virus) and AIDS (HIV-1) necessitate urgent attention to develop diagnostic protocols and assays for rapid detection and screening. Rapid and accurate detection of first cases with certainty will contribute significantly in preventing disease transmission and escalation to pandemic levels. As a result, there is a need to develop technologies that can meet the heavy demand of an all-embedded, inexpensive, specific and fast biosensing for the detection and screening of pathogens in active or latent forms to offer quick diagnosis and early treatments in order to avoid disease aggravation and unnecessary late treatment costs. Nucleic acid aptamers are short, single-stranded RNA or DNA sequences that can selectively bind to specific cellular and biomolecular targets. Aptamers, as new-age bioaffinity probes, have the necessary biophysical characteristics for improved pathogen detection. This article seeks to review global pandemic situations in relation to advances in pathogen detection systems. It particularly discusses aptameric biosensing and establishes application opportunities for effective pandemic monitoring. Insights into the application of continuous polymeric supports as the synthetic base for aptamer coupling to provide the needed convective mass transport for rapid screening is also presented.


Assuntos
Aptâmeros de Nucleotídeos , Técnicas Biossensoriais , Pandemias , Bactérias/genética , Humanos , Vírus/genética
4.
Prep Biochem Biotechnol ; 46(7): 657-65, 2016 Oct 02.
Artigo em Inglês | MEDLINE | ID: mdl-26759918

RESUMO

Pedobacter cryoconitis BG5 are psychrophiles isolated from the cold environment and capable of proliferating and growing well at low temperature regime. Their cellular products have found a broad spectrum of applications, including in food, medicine, and bioremediation. Therefore, it is imperative to develop a high-cell density cultivation strategy coupled with optimized growth medium for P. cryoconitis BG5. To date, there has been no published report on the design and optimization of growth medium for P. cryoconitis, hence the objective of this research project. A preliminary screening of four commercially available media, namely tryptic soy broth, R2A, Luria Bertani broth, and nutrient broth, was conducted to formulate the basal medium. Based on the preliminary screening, tryptone, glucose, NaCl, and K2HPO4 along with three additional nutrients (yeast extract, MgSO4, and NH4Cl) were identified to form the basal medium which was further analyzed by Plackett-Burman experimental design. Central composite experimental design using response surface methodology was adopted to optimize tryptone, yeast extract, and NH4Cl concentrations in the formulated growth medium. Statistical data analysis showed a high regression factor of 0.84 with a predicted optimum optical (600 nm) cell density of 7.5 using 23.7 g/L of tryptone, 8.8 g/L of yeast extract, and 0.7 g/L of NH4Cl. The optimized medium for P. cryoconitis BG5 was tested, and the observed optical density was 7.8. The cost-effectiveness of the optimized medium was determined as 6.25 unit prices per gram of cell produced in a 250-ml Erlenmeyer flask.


Assuntos
Pedobacter/crescimento & desenvolvimento , Custos e Análise de Custo , Meios de Cultura , Modelos Teóricos
5.
J Sep Sci ; 37(5): 455-64, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24376196

RESUMO

To date, the number of published reports on the large-volume preparation of polymer-based monolithic chromatography adsorbents is still lacking and is of great importance. Many critical factors need to be considered when manufacturing a large-volume polymer-based monolith for chromatographic applications. Structural integrity, validity, and repeatability are thought to be the key factors determining the usability of a large-volume monolith in a separation process. In this review, we focus on problems and solutions pertaining to heat dissipation, pore size distribution, "wall channel" effect, and mechanical strength in monolith preparation. A template-based method comprising sacrificial and nonsacrificial techniques is possibly the method of choice due to its precise control over the porous structure. However, additional expensive steps are usually required for the template removal. Other strategies in monolith preparation are also discussed.


Assuntos
Cromatografia/instrumentação , Polímeros/química , Absorção , Animais , Cromatografia/métodos , DNA/química , DNA/isolamento & purificação , Humanos , Polímeros/síntese química , Proteínas/química , Proteínas/isolamento & purificação
6.
J Sep Sci ; 35(22): 3208-16, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23001922

RESUMO

Endotoxin lipopolysaccharide removal from plasmid DNA-based vaccine remains a very challenging task for bioprocess engineers. This paper examined the potential use and advantages of divalent cation (Zn(2+), Ca(2+), Mg(2+)) induced aggregation as a plasmid DNA purification method for lipopolysaccharide removal. Analysis of zeta potential, hydrodynamic size, percentage of aggregation; UV-Vis spectroscopy and electron microscopy were performed to determine the optimal cation for preferential aggregation of lipopolysaccharide over plasmid DNA. The results from the hydrodynamic size analysis showed that the addition of Zn(2+) resulted in the maximum theoretical number of lipopolysaccharide molecules per aggregate particle. Dynamic light scattering analysis showed that plasmid DNA aggregates formed a larger maximum hydrodynamic size when it was treated with Ca(2+) than the other two cations. The K(m) value for lipopolysaccharide-Zn(2+) was substantially low (0.28 M) and considerably large (>2 M) for plasmid DNA-Zn(2+). Scatchard plots for plasmid DNA cations showed positive slopes indicating that there was a minimum concentration of plasmid DNA or cations before a significant aggregation occurred. This work concluded that Zn(2+) had the most preferential aggregative interaction with lipopolysaccharide compared to Mg(2+) and Ca(2+).


Assuntos
Bioquímica/métodos , Cálcio/química , DNA/isolamento & purificação , Endotoxinas/química , Lipopolissacarídeos/química , Magnésio/química , Plasmídeos/isolamento & purificação , Zinco/química , Adsorção , Cátions Bivalentes/química , DNA/química , Contaminação por DNA , Cinética , Plasmídeos/química
7.
Anal Chem ; 83(1): 391-7, 2011 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-21117617

RESUMO

Recent advancements in plasmid DNA (pDNA) production involve the development of innovative and cost-effective methods as well as reduced number of unit operations. This study investigates the feasibilities of using a metal salt to selectively remove endotoxins from clarified cell lysates containing plasmid DNA. Screening of endotoxin precipitation in various metal salt solutions and optimization of process conditions (pH, ion concentration, temperature, and incubation time) using central composite design experiments have been carried out successfully. Results show that selective endotoxin precipitation (<0.05 EU/µg) can economically be carried out during the alkaline cell lysis procedure (neutralization step) at a pH condition similar to that of alkaline-lysed cell lysate, a low ZnSO(4) concentration (0.5 M), a minimum incubation time (30 min), and a temperature of 15 °C. In summary, this method provides ease of subsequent plasmid DNA purification due to reduced bulk impurities and cost-effective and most importantly high endotoxin removal (>80%) and plasmid recovery (>90%).


Assuntos
Precipitação Química , DNA/isolamento & purificação , Endotoxinas/isolamento & purificação , Caranguejos Ferradura , Proteínas de Membrana/metabolismo , Metais/química , Plasmídeos/isolamento & purificação , Animais , DNA/química , Endotoxinas/química , Endotoxinas/metabolismo , Concentração de Íons de Hidrogênio , Plasmídeos/química , Sais/química , Temperatura , Fatores de Tempo , Sulfato de Zinco/química
8.
Crit Rev Biotechnol ; 31(1): 32-52, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-20879832

RESUMO

The exponentially growing human population and the emergence of new diseases are clear indications that the world can no longer depend solely on conventional vaccine technologies and production schemes. The race to find a new vaccine technology is crucial to help speed up and complement the World Health Organization (WHO) disease elimination program. The ultimate goal is to uncover fast and efficient production schemes in the event of a pandemic, and also to effectively fight deadly diseases such as malaria, bird flu, hepatitis, and human immunodeficiency virus (HIV). Plasmid DNA vaccines, if properly formulated, offer specific priming of the immune system and similar or even better prophylactic effects than conventional vaccines. This article discusses many of the critical issues that need to be considered when developing fast, effective, and reliable plasmid DNA vaccine manufacturing processes. Different modes of plasmid production via bacterial fermentation are compared. Plasmid purification by chromatography is specifically discussed as it is the most commercially viable bioprocess engineering technique for continuous purification of supercoiled plasmid DNA. Current techniques and progress covering the area of plasmid DNA vaccine design, formulation, and delivery are also put forward.


Assuntos
Biotecnologia/métodos , Sistemas de Liberação de Medicamentos , Plasmídeos , Vacinas de DNA , Animais , DNA Super-Helicoidal/genética , Desenho de Fármacos , Humanos , Plasmídeos/genética , Plasmídeos/metabolismo , Vacinas de DNA/administração & dosagem
9.
Microb Cell Fact ; 10: 16, 2011 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-21375765

RESUMO

BACKGROUND: Plasmid-based measles vaccines offer great promises over the conventional fertilised egg method such as ease of manufacture and mimic wild-type intracellular antigen expression. The increasing number of clinical trials on plasmid-based measles vaccines has triggered the need to make more in less time. RESULTS: In this work, we investigated the process variables necessary to improve the volumetric and specific yields of a model plasmid-based measles vaccine (pcDNA3F) harboured in E. coli DH5α. Results from growth medium optimisation in 500 mL shake flasks by response surface methodology (RSM) generated a maximum volumetric yield of 13.65 mg/L which was 1.75 folds higher than that of the base medium. A controlled fed-batch fermentation employing strategic glycerol feeding and optimised growth conditions resulted in a remarkable pcDNA3F volumetric yield of 110 mg/L and a specific yield of 14 mg/g. In addition, growth pH modification and temperature fluctuation between 35 and 45°C were successfully employed to improve plasmid production. CONCLUSION: Production of a high copy number plasmid DNA containing a foreign gene of interest is often hampered by the low plasmid volumetric yield which results from the over expression of foreign proteins and metabolic repressors. In this work, a simple bioprocess framework was employed and successfully improved the production of pcDNA3F.


Assuntos
Escherichia coli/crescimento & desenvolvimento , Vacina contra Sarampo/biossíntese , Meios de Cultura/química , Meios de Cultura/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Concentração de Íons de Hidrogênio , Vacina contra Sarampo/genética , Vacina contra Sarampo/imunologia , Plasmídeos/química , Plasmídeos/metabolismo , Temperatura , Vacinas de DNA/biossíntese , Vacinas de DNA/química , Vacinas de DNA/imunologia
10.
Materials (Basel) ; 14(23)2021 Nov 25.
Artigo em Inglês | MEDLINE | ID: mdl-34885320

RESUMO

Porous monoliths prepared using templates are highly sought after for filtration applications due to their good mass transport properties and high permeability. Current templates, however, often lead to the formation of dead-end pores and irregular pore distributions, which reduce the efficiency of the substrate flow across the monolith column. This study focused on the preparation of a microsphere-templated porous monolith for wastewater filtration. The optimal template/monomer ratio (50:50, 60:40, 70:30) was determined, and appropriate template removal techniques were assessed for the formation of homogenous pores. The physicochemical characteristics and pore homogeneity of the monoliths were examined. The 60:40 ratio was determined to result in monoliths with homogeneous pore distributions ranging from 1.9 µm to 2.3 µm. SEM and FTIR investigations revealed that solvent treatment was effective for removing templates from the resulting solid monolith. The water quality assessments revealed reductions in the turbidity and the total number of suspended particles in the tested wastewater of up to 96-99%. The findings of this study provide insightful knowledge regarding the fabrication of monoliths with homogenous pores that are beneficial for wastewater treatment.

11.
Polymers (Basel) ; 13(21)2021 Oct 22.
Artigo em Inglês | MEDLINE | ID: mdl-34771196

RESUMO

Monolith is an emerging technology applicable for separation, filtration, and chromatography due to its interconnected pore structure. However, the current templates used to form monolith pores are associated with poor heat dissipation, uneven pore size distribution, and relatively low mechanical strength during monolith scale-up. Templates made from polymeric microsphere particles were synthesized via a solvent evaporation technique using different types of polymer (polystyrene, polycaprolactone, polypropylene, polyethylene, and poly (vinyl-alcohol) at varied polymer (10-40 wt%) and surfactant (5-10%) concentrations. The resulting microsphere particles were tested as a monolith template for the formation of homogenous pores. Among the tested polymers, polystyrene at 10 wt% concentration demonstrated good particle morphology determined to around 1.94-3.45 µm. The addition of surfactant at a concentration of 7-10 wt% during microsphere synthesis resulted in the formation of well-shaped and non-aggregating microsphere particles. In addition, the template has contributed to the production of porous monoliths with enhanced thermal stability. The thermogravimetric analysis (TGA) indicated monolith degradation between 230 °C and 450 °C, implying the material excellent mechanical strength. The findings of the study provide insightful knowledge on the feasibility of polymeric microsphere particles as a pore-directing template to fabricate monoliths with desired pore structures.

12.
J Sep Sci ; 32(15-16): 2485-94, 2009 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-19603394

RESUMO

Polymethacrylate monoliths, specifically poly(glycidyl methacrylate-co-ethylene dimethacrylate) or poly(GMA-co-EDMA) monoliths, are a new generation of chromatographic supports and are significantly different from conventional particle-based adsorbents, membranes, and other monolithic supports for biomolecule purification. Similar to other monoliths, polymethacrylate monoliths possess large pores which allow convective flow of mobile phase and result in high flow rates at reduced pressure drop, unlike particulate supports. The simplicity of the adsorbent synthesis, pH resistance, and the ease and flexibility of tailoring their pore size to that of the target biomolecule are the key properties which differentiate polymethacrylate monoliths from other monoliths. Polymethacrylate monoliths are endowed with reactive epoxy groups for easy functionalization (with anion-exchange, hydrophobic, and affinity ligands) and high ligand retention. In this review, the structure and performance of polymethacrylate monoliths for chromatographic purification of biomolecules are evaluated and compared to those of other supports. The development and use of polymethacrylate monoliths for research applications have grown rapidly in recent times and have enabled the achievement of high through-put biomolecule purification on semi-preparative and preparative scales.


Assuntos
Cromatografia/instrumentação , Ácidos Polimetacrílicos/química , Adsorção , Cromatografia/métodos , Matemática , Ácidos Nucleicos/isolamento & purificação , Oligonucleotídeos/isolamento & purificação , Peptídeos/isolamento & purificação , Porosidade , Pressão , Proteínas/isolamento & purificação , Propriedades de Superfície , Vírion/isolamento & purificação
13.
Sci Rep ; 9(1): 14501, 2019 10 10.
Artigo em Inglês | MEDLINE | ID: mdl-31601836

RESUMO

Immobilisation of aptameric ligands on solid stationary supports for effective binding of target molecules requires understanding of the relationship between aptamer-polymer interactions and the conditions governing the mass transfer of the binding process. Herein, key process parameters affecting the molecular anchoring of a thrombin-binding aptamer (TBA) onto polymethacrylate monolith pore surface, and the binding characteristics of the resulting macroporous aptasensor were investigated. Molecular dynamics (MD) simulations of the TBA-thrombin binding indicated enhanced Guanine 4 (G4) structural stability of TBA upon interaction with thrombin in an ionic environment. Fourier-transform infrared spectroscopy and thermogravimetric analyses were used to characterise the available functional groups and thermo-molecular stability of the immobilised polymer generated with Schiff-base activation and immobilisation scheme. The initial degradation temperature of the polymethacrylate stationary support increased with each step of the Schiff-base process: poly(Ethylene glycol Dimethacrylate-co-Glycidyl methacrylate) or poly(EDMA-co-GMA) [196.0 °C (±1.8)]; poly(EDMA-co-GMA)-Ethylenediamine [235.9 °C (±6.1)]; poly(EDMA-co-GMA)-Ethylenediamine-Glutaraldehyde [255.4 °C (±2.7)]; and aptamer-modified monolith [273.7 °C (±2.5)]. These initial temperature increments reflected in the associated endothermic energies were determined with differential scanning calorimetry. The aptameric ligand density obtained after immobilisation was 480 pmol/µL. Increase in pH and ionic concentration affected the surface charge distribution and the binding characteristics of the aptamer-modified disk-monoliths, resulting in the optimum binding pH and ionic concentration of 8.0 and 5 mM Mg2+, respectively. These results are critical in understanding and setting parametric constraints indispensable to develop and enhance the performance of aptasensors.

14.
Biochimie ; 157: 204-212, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30513369

RESUMO

Biocarriers are pivotal in enhancing the reusability of biocatalyst that would otherwise be less economical for industrial application. Ever since the induction of enzymatic technology, varied materials have been assessed for their biocompatibility with enzymes of distinct functionalities. Herein, cellulase was immobilized onto polymethacrylate particles (ICP) as the biocarrier grafted with ethylenediamine (EDA) and glutaraldehyde (GA). Carboxymethyl cellulose (CMC) was used as a model substrate for activity assay. Enzyme immobilization loading was determined by quantifying the dry weight differential of ICP (pre-& post-immobilization). Cellulase was successfully demonstrated to be anchored upon ICP and validated by FTIR spectra analysis. The optimal condition for cellulase immobilization was determined to be pH 6 at 20 °C. The maximum CMCase activity was achieved at pH 5 and 50 °C. Residual activity of ∼50% was retained after three iterations and dipped to ∼18% on following cycle. Also, ICP displayed superior pH adaptability as compared to free cellulase. The specific activity of ICP was 65.14 ±â€¯1.11% relative to similar amount of free cellulase.


Assuntos
Carboximetilcelulose Sódica/química , Celulase/química , Enzimas Imobilizadas/química , Ácidos Polimetacrílicos/química , Estabilidade Enzimática , Temperatura Alta , Concentração de Íons de Hidrogênio , Hidrólise
15.
Curr Drug Targets ; 19(3): 248-258, 2018 02 08.
Artigo em Inglês | MEDLINE | ID: mdl-27321771

RESUMO

BACKGROUND: The search for smart delivery systems for enhanced pre-clinical and clinical pharmaceutical delivery and cell targeting continues to be a major biomedical research endeavor owing to differences in the physicochemical characteristics and physiological effects of drug molecules, and this affects the delivery mechanisms to elicit maximum therapeutic effects. Targeted drug delivery is a smart evolution essential to address major challenges associated with conventional drug delivery systems. These challenges mostly result in poor pharmacokinetics due to the inability of the active pharmaceutical ingredients to specifically act on malignant cells thus, causing poor therapeutic index and toxicity to surrounding normal cells. Aptamers are oligonucleotides with engineered affinities to bind specifically to their cognate targets. Aptamers have gained significant interests as effective targeting elements for enhanced therapeutic delivery as they can be generated to specifically bind to wide range of targets including proteins, peptides, ions, cells and tissues. Notwithstanding, effective delivery of aptamers as therapeutic vehicles is challenged by cell membrane electrostatic repulsion, endonuclease degradation, low pH cleavage, and binding conformation stability. OBJECTIVE: The application of molecularly engineered biodegradable and biocompatible polymeric particles with tunable features such as surface area and chemistry, particulate size distribution and toxicity creates opportunities to develop smart aptamer-mediated delivery systems for controlled drug release. RESULTS: This article discusses opportunities for particulate aptamer-drug formulations to advance current drug delivery modalities by navigating active ingredients through cellular and biomolecular traffic to target sites for sustained and controlled release at effective therapeutic dosages while minimizing systemic cytotoxic effects. CONCLUSION: A proposal for a novel drug-polymer-aptamer-polymer (DPAP) design of aptamer-drug formulation with stage-wise delivery mechanism is presented to illustrate the potential efficacy of aptamer- polymer cargos for enhanced cell targeting and drug delivery.


Assuntos
Antineoplásicos/administração & dosagem , Aptâmeros de Nucleotídeos/síntese química , Polímeros/síntese química , Antineoplásicos/química , Antineoplásicos/uso terapêutico , Aptâmeros de Nucleotídeos/administração & dosagem , Aptâmeros de Nucleotídeos/química , Portadores de Fármacos/administração & dosagem , Portadores de Fármacos/síntese química , Portadores de Fármacos/química , Sistemas de Liberação de Medicamentos , Humanos , Concentração de Íons de Hidrogênio , Neoplasias/tratamento farmacológico , Polímeros/administração & dosagem , Polímeros/química
16.
Biotechnol Prog ; 34(1): 249-261, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-28699244

RESUMO

Targeted delivery of drug molecules to specific cells in mammalian systems demonstrates a great potential to enhance the efficacy of current pharmaceutical therapies. Conventional strategies for pharmaceutical delivery are often associated with poor therapeutic indices and high systemic cytotoxicity, and this result in poor disease suppression, low surviving rates, and potential contraindication of drug formulation. The emergence of aptamers has elicited new research interests into enhanced targeted drug delivery due to their unique characteristics as targeting elements. Aptamers can be engineered to bind to their cognate cellular targets with high affinity and specificity, and this is important to navigate active drug molecules and deliver sufficient dosage to targeted malignant cells. However, the targeting performance of aptamers can be impacted by several factors including endonuclease-mediated degradation, rapid renal filtration, biochemical complexation, and cell membrane electrostatic repulsion. This has subsequently led to the development of smart aptamer-immobilized biopolymer systems as delivery vehicles for controlled and sustained drug release to specific cells at effective therapeutic dosage and minimal systemic cytotoxicity. This article reports the synthesis and in vitro characterization of a novel multi-layer co-polymeric targeted drug delivery system based on drug-loaded PLGA-Aptamer-PEI (DPAP) formulation with a stage-wise delivery mechanism. A thrombin-specific DNA aptamer was used to develop the DPAP system while Bovine Serum Albumin (BSA) was used as a biopharmaceutical drug in the synthesis process by ultrasonication. Biophysical characterization of the DPAP system showed a spherical shaped particulate formulation with a unimodal particle size distribution of average size ∼0.685 µm and a zeta potential of +0.82 mV. The DPAP formulation showed a high encapsulation efficiency of 89.4 ± 3.6%, a loading capacity of 17.89 ± 0.72 mg BSA protein/100 mg PLGA polymeric particles, low cytotoxicity and a controlled drug release characteristics in 43 days. The results demonstrate a great promise in the development of DPAP formulation for enhanced in vivo cell targeting. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:249-261, 2018.


Assuntos
Aptâmeros de Nucleotídeos/química , Fenômenos Biofísicos , Sistemas de Liberação de Medicamentos , Nanopartículas/química , Animais , Aptâmeros de Nucleotídeos/uso terapêutico , Movimento Celular/efeitos dos fármacos , Humanos , Iminas/química , Iminas/uso terapêutico , Nanopartículas/uso terapêutico , Tamanho da Partícula , Polietilenos/química , Polietilenos/uso terapêutico , Soroalbumina Bovina/química
17.
J Biol Methods ; 4(2): e71, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-31453229

RESUMO

Endotoxin has been one of the topical chemical contaminants of major concern to researchers, especially in the field of bioprocessing. This major concern of researchers stems from the fact that the presence of Gram-negative bacterial endotoxin in intracellular products is unavoidable and requires complex downstream purification steps. For instance, endotoxin interacts with recombinant proteins, peptides, antibodies and aptamers and these interactions have formed the foundation for most biosensors for endotoxin detection. It has become imperative for researchers to engineer reliable means/techniques to detect, separate and remove endotoxin, without compromising the quality and quantity of the end-product. However, the underlying mechanism involved during endotoxin-biomolecule interaction is still a gray area. The use of quantitative molecular microscopy that provides high resolution of biomolecules is highly promising, hence, may lead to the development of improved endotoxin detection strategies in biomolecule preparation. Förster resonance energy transfer (FRET) spectroscopy is one of the emerging most powerful tools compatible with most super-resolution techniques for the analysis of molecular interactions. However, the scope of FRET has not been well-exploited in the analysis of endotoxin-biomolecule interaction. This article reviews endotoxin, its pathophysiological consequences and the interaction with biomolecules. Herein, we outline the common potential ways of using FRET to extend the current understanding of endotoxin-biomolecule interaction with the inference that a detailed understanding of the interaction is a prerequisite for the design of strategies for endotoxin identification and removal from protein milieus.

18.
Eur J Pharm Sci ; 96: 8-19, 2017 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-27593990

RESUMO

Cancer is a leading cause of global mortality. Whilst anticancer awareness programs have increased significantly over the years, scientific research into the development of efficient and specific drugs to target cancerous cells for enhanced therapeutic effects has not received much clinical success. Chemotherapeutic agents are incapable of acting specifically on cancerous cells, thus causing low therapeutic effects accompanied by toxicity to surrounding normal tissues. The search for smart, highly specific and efficient cancer treatments and delivery systems continues to be a significant research endeavor. Targeted cancer therapy is an evolving treatment approach with great promise in enhancing the efficacy of cancer therapies via the delivery of therapeutic agents specifically to and into desired tumor cells using viral or non-viral targeting elements. Viral oncotherapy is an advanced cancer therapy based on the use of oncolytic viruses (OV) as elements to specifically target, replicate and kill malignant cancer cells selectively without affecting surrounding healthy cells. Aptamers, on the other hand, are non-viral targeting elements that are single-stranded nucleic acids with high specificity, selectivity and binding affinity towards their cognate targets. Aptamers have emerged as a new class of bioaffinity targeting elements can be generated and molecularly engineered to selectively bind to diverse targets including proteins, cells and tissues. This article discusses, comparatively, the potentials and impacts of both viral and aptamer-mediated targeted cancer therapies in advancing conventional drug delivery systems through enhanced target specificity, therapeutic payload, bioavailability of the therapeutic agents at the target sites whilst minimizing systemic cytotoxicity. This article emphasizes on effective site-directed targeting mechanisms and efficacy issues that impact on clinical applications.


Assuntos
Aptâmeros de Nucleotídeos/administração & dosagem , Sistemas de Liberação de Medicamentos , Neoplasias/terapia , Vírus Oncolíticos , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/uso terapêutico , Aptâmeros de Nucleotídeos/uso terapêutico , Humanos , Polímeros/administração & dosagem , Polímeros/uso terapêutico
19.
J Chromatogr B Analyt Technol Biomed Life Sci ; 1015-1016: 121-134, 2016 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-26919447

RESUMO

Biomedical research advances over the past two decades in bioseparation science and engineering have led to the development of new adsorbent systems called monoliths, mostly as stationary supports for liquid chromatography (LC) applications. They are acknowledged to offer better mass transfer hydrodynamics than their particulate counterparts. Also, their architectural and morphological traits can be tailored in situ to meet the hydrodynamic size of molecules which include proteins, pDNA, cells and viral targets. This has enabled their development for a plethora of enhanced bioscreening applications including biosensing, biomolecular purification, concentration and separation, achieved through the introduction of specific functional moieties or ligands (such as triethylamine, N,N-dimethyl-N-dodecylamine, antibodies, enzymes and aptamers) into the molecular architecture of monoliths. Notwithstanding, the application of monoliths presents major material and bioprocess challenges. The relationship between in-process polymerisation characteristics and the physicochemical properties of monolith is critical to optimise chromatographic performance. There is also a need to develop theoretical models for non-invasive analyses and predictions. This review article therefore discusses in-process analytical conditions, functionalisation chemistries and ligands relevant to establish the characteristics of monoliths in order to facilitate a wide range of enhanced bioscreening applications. It gives emphasis to the development of functional polymethacrylate monoliths for microfluidic and preparative scale bio-applications.


Assuntos
Cromatografia Líquida , Polímeros , Anticorpos/isolamento & purificação , Separação Celular , Cromatografia Líquida/instrumentação , Cromatografia Líquida/métodos , Cromatografia Líquida/tendências , Humanos , Proteínas/isolamento & purificação , Vírus/isolamento & purificação
20.
Crit Rev Anal Chem ; 46(6): 521-37, 2016 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26980177

RESUMO

The quest to improve the detection of biomolecules and cells in health and life sciences has led to the discovery and characterization of various affinity bioprobes. Libraries of synthetic oligonucleotides (ssDNA/ssRNA) with randomized sequences are employed during Systematic Evolution of Ligands by Exponential Enrichment (SELEX) to select highly specific affinity probes called aptamers. With much focus on the generation of aptamers for a variety of target molecules, conventional SELEX protocols have been modified to develop new and improved SELEX protocols yielding highly specific and stable aptamers. Various techniques have been used to analyze the binding interactions between aptamers and their cognate molecules with associated merits and limitations. This article comprehensively reviews research advancements in the generation of aptamers, analyses physicochemical conditions affecting their binding characteristics to cellular and biomolecular targets, and discusses various field applications of aptameric binding. Biophysical techniques employed in the characterization of the molecular and binding features of aptamers to their cognate targets are also discussed.


Assuntos
Aptâmeros de Nucleotídeos/química , Técnica de Seleção de Aptâmeros/métodos , Sítios de Ligação , Ligantes
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