BMe Research Grant


Wagner István

email address


BMe Research Grant - 2013

George A. Oláh Doctoral School of Chemistry and Chemical Technology  

Department of Organic Chemistry and Technology

Supervisor: MAROSI György

Solid Formulation of Biodrugs by Electrospinning 

Introducing the research area

The growing importance of APIs (active pharmaceutical ingredients) of biotechnological origin means huge challenge for pharmaceutical technologists. These biodrugs are highly sensitive to environmental factors, thus traditional drying technologies (lyophilization spray drying) enable their solid formulation only with significant loss of their biological activity and high costs because these techniques have severe time and energy demand. In this research, we investigate the possible application of the electrospinning technology, widespread in plastic and textile industry but not yet applied in pharmaceutical industry, to replace classic drying techniques for the solid formulation of biodrugs. 

Brief introduction of the research place

The Research Group of Safety, Environmental and Pharmaceutical Technologies (SAFEPHARMATECH) is committed to technology and materials science related researches that are aiming to improve the quality of life in a non-sector specific way. This includes the improvement of safe, controlled, continuous and integrated technologies regarding the field of pharmaceutical industry, the researches for fire safety and the use of renewable energy sources in the field of automotive industry and the complex recycling of plastic waste in the field of environmental protection.

History and context of the research

The second half of the 20th century had seen huge advances in the field of biotechnology and the tendency continued unabated in the 21st century broadening the pharmaceutical palette with various APIs (active pharmaceutical ingredients) of biotechnological origin. The broad spectrum of biotechnology based API-s or biodrugs includes proteins, peptids, whole cells, nucleic acids, viral particles and vaccines [1], all being produced in a biotechnological way where the end product, the biodrug itself is usually in a water solution or suspension. Typical features of biodrugs are high sensitivity to their physical and chemical environment (temperature, pressure, mechanical stress, pH, ion concentration etc.) and low stability in aqueous medium [2]. The gentle removal of water from these systems significantly increases the stability of the biodrugs thus providing improved transportability and shelf life.

The most usual way of water removal is lyophilization [3] that may decrease the biological activity through the irreversible damaging of the structure of the biodrug by water crystals [4]. Another disadvantage of lyophilization is that it is time-consuming and has high energy demand [5, 6]. Spray drying is a less widespread alternative where drying is provided by heated air thus also decreasing biological activity of biodrugs usually having low thermal stability.

The growing expansion of biodrugs and their solid drug forms in pharmaceutical development means new challenges for pharmaceutical technologies and among all these the greatest demand for technological improvement is in the field of drying.

The research goal, open questions

In our researches, two widely used biological APIs have been chosen for formulation experiments: the first one is Lactobacillus acidophilus, a probiotic bacterium that is an important member of human microflora. Besides its numerous therapeutic application, recently it has been successfully used in the treatment of the most common gynecological illness, the bacterial vaginosis (BV) [7]. The other biodrug was beta-galactosidase, an enzyme produced by the small intestine that catalyzes the hydrolysis of lactose into glucose and galactose. The absence of adequate quantity of this enzyme causes the symptoms of lactose intolerance (nausea, colic, distention and diarrhea) in which 75% of the adult population are involved [8]. Not only both biodrugs have high therapeutic importance but they also serve as good models of the formulation experiments of the most widely used biodrugs, the probiotics and the protein APIs, medicines of diseases untreatable so far.

The aim of our research is to develop an alternative drying method instead of lyophilization that is fast, productive, continuous, has a low energy demand and is gentle enough to enable the production of solid biodrug formula.

In the case of both biodrugs, we have investigated the applicability of electrospinning, a new technology in pharmaceutical researches to produce solid drug formula from aqueous solution with the lowest loss of activity and the highest stability of the biodrug.



Lactobacillus acidophilus was fermented in MRS Broth at 37°C for two days and then centrifuged. To the suspension being obtained this way, typical excipients of the bacterium drying such as trehalose, saccharose and skim milk powder were added. Beta-galactosidase was kindly provided by Optiferm and the concentration of the initial solution was 0.25 g/ml. The aqueous suspension or solution of the biodrug was added to the polymer solutions of which the electrospinning was carried out.

Electrospinning is a simple and effective technology to produce very fine polymer fibers with a diameter of 50-1000 nm. PVA (poly-vinyl-alcohol) and PVP (poly-vinyl-pyrrolidone) were used as polymer matrices, both accepted by drug administration authorities. The solutions containing the polymer and the biodrug were attached to a high voltage (35 kV) loaded electrode which was connected to a spinneret and a grounded collector electrode. Due to the high electric field, the liquid gains conical shape that is called Taylor-cone from which thin jet can emerge to the grounded collector as long as the Coulomb repulsion of the charges on the cone surface exceed the retaining force of the surface tension. The emerging liquid jet is drawn by electrostatic forces, gets thinner, grows large surface causing a fast evaporation of the solvent. The fiber-shaped solid polymer can be collected and removed from the collector. The drying process, the production of the dry fibers from aqueous solution takes less than a second. [9] Dry samples were stored at 7°C in sterile closed vessels.  




Figure 1: Solid Formulation of Biodrugs by Electrospinning


The morphology of the fibers was investigated with scanning electron microscopy (SEM). In order to gain accurate results about the indulgence of the technology, enzyme activity and the number of colony forming units were determined in the initial aqueous solutions and the resulting polymer fibers.

Bacterial suspensions and solid products were added gravimetrically and then diluted with sterile water and dilution series were prepared. The diluted suspensions were added to MRS agar plates and incubated for 48 hours at 37°C under anaerob circumstances in the presence of oxygen binding material (Anaerocult), and then the number of colony forming units was counted.

The activity of beta-galactosidase was investigated by the tracking of catalyzed reactions carried out at 55°C, pH 4,6 for 10 minutes with continuous stirring and stopped with 1 M sodium-carbonate solution. Two substrates were tested: orto-nitrophenyl-beta-galactoside and lactose. The former is hydrolized by the enzyme into galactose and orto-nitrophenol that gives a yellow color in alkali solution thus it can be determined with UV-VIS spectrophotometry. The latter is natural substrate of the enzyme where the products – glucose and galactose – were quantified with HPLC (high performance liquid chromatography).        


In our researches we have successfully produced polymer fibers containing biodrugs. In the cases of both fiber forming polymers, the polymer was wrapping the bacteria as a coating while the enzyme containing polymers showed smaller knobs (Figure 2).  In several cases, bacteria stuck together can be observed suggesting that the forces acting during the fiber production are less than necessary for the separation of bacteria stuck together during cell division. This implies that the bacteria are exposed merely to a modest mechanical stress during the drying process.



Figure 2: Biodrugs embedded into polymer fibers (A: Lactobacillus acidophilus in PVA, B: Lactobacillus acidophilus in PVP, C: beta-galactosidase in PVP)


Our SEM results show that the polymer fibers containing biodrugs do not have larger diameter than placebo polymer fibers except those parts where the cells are wrapped by the matrix, indicating that the electrospinning process itself is not influenced by the presence of biodrugs. This is promising from the aspect of scale-up possibilities because the scaling up of PVA and PVP nanofiber production technologies are thoroughly investigated [10, 11].

Considering our aims, the most important result was that the biological activity of the biodrugs – the viability of the bacteria and the activity of the enzyme – do not decrease significantly during the whole process. This means that we have succeeded to produce solid polymer nanoweb containing biodrug with a bioactivity nearly 100% compared to that of the initial solvents.

As preserving their effectiveness is a fundamental requirement to drugs, we have investigated how the activity of biodrugs changes in the fibers. After one year, 1 mg of Lactobacillus acidophilus containing sample contained more than 1 million active bacteria, thus the effective dose. Remarkable is that after one year, the number of living bacteria decreased only to its third while other papers dealing with lyophilization [4, 12] reported a decrease of one or more orders of magnitude during less than a year. These exceptionally good results might be attributed to the gentle milieu of drying and the protective shield of polymer formed around the bacteria during the spinning. The activity of the enzymes did not decrease either in a one year period and in this case about 20 mg sample contained the effective dose. So the biodrugs contained in the polymer fibers produced in these experiments were capable of preserving their biological activity in the long term. Our results indicate that electrospinning can be a real alternative for traditional drying techniques.


Expected impact and further research

The results of our experiments show that electrospinning enables producing stable solid biodrugs contained in polymer fibers in a much more gentle way than with traditional drying processes. Scale-up experiments are already in progress. Nowadays, the production of 10 kg PVA fiber per day is possible, thus if the scaling up causes no significant loss in the activity, this technology is capable of producing notable amount of biodrugs containing effective doses. Furthermore, we are planning to investigate the testing of other biodrugs also representing high therapeutic importance, especially protein medicines that have a growing importance (e.g. monoclonal antibodies).


Publications, references, links



I. I. Wagner, Nagy Z. K., Á. Suhajda, T. Tobak, A. H. Harasztos, H. Pataki, G. Marosi, Solid Dosage Form of Living Bacteria Prepared by Electrospinning. New Biotechnology. (2013) if.: 1,706 (submitted)


II. I. Wagner, H. Pataki, A. Balogh, Z.K. Nagy, A. H. Harasztos, Á. Suhajda, G. Marosi, Electrospun nanofibers for topical drug delivery. European Journal of Pharmaceutical Sciences, 44, Suppl. 1: 148 (2011) if: 3,291


III. Z. K. Nagy, A. Balogh, I. Wagner, P. Sóti, H. Pataki, K. Molnár, G. Marosi, Nanofibrous drug delivery systems for enhanced dissolution prepared by electrospinning. European Journal of Pharmaceutical Sciences, 44, Suppl. 1: 152-153 (2011) if: 3,291


IV. Z. K. Nagy, K. Nyúl, I. Wagner, K. Molnár, G. Marosi, Electrospun water soluble polymer mat for ultrafast release of Donepezil HCl. Express Polymer Letters 4: 763–772 (2010) if: 1,575





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[8] D.M. Paige, Lactose Intolerance, Encyclopedia of Human Nutrition (Third Edition). Pages 67–73 (2013)


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[12] G. Zayed, Y. H. Roos, Influence of trehalose and moisture content on survival of Lactobacillus salivarius subjected to freeze-drying and storage. Process Biochemistry 39: 1081-1086