polymer materials against the microorganism's attack

Polymer materials against the microorganism’s attack

Yu. Savelyev

National Academy of Sciences of Ukraine, Institute of Macromolecular Chemistry, Department of Chemistry of Heterochain Polymers&Interpenetrating Polymer Network, 48, Kharkovskoe shosse, Kiev, 02160, Ukraine

Many polymers are susceptible to attack of microorganisms in the environment resulting in product failure. Theoretically it could be assumed that synthetic materials would be inaccessible for known destructors, since similar compounds did not exist in a nature and adapted to them microorganisms were absent. However, in spite of apparent primitivity of organization, microorganisms were not isolated from environmental influence. The most active biodestructors, specialized on polymers, belongs to the genus: Aspergillis and Penicillium. They are also known as contaminates of various materials, and accordingly to their action on human organism they relay to the potential dangerous group, defined based on the ”level of biological hazard”, as provocateurs of mycosis, allergic states and opportunistic human diseases. Actually processes of biodegradation, induced by micromycets and its products of methabolism, including toxins are also considered in medic-sanitary aspect. Novel polymeric biologically active fungistatic (antimycotic) and antimicrobial materials for human, animals and materials protection against microorganism attack have been synthesized. Polymers’ structure-properties relationships have been studied. Structure-chemical and physical modification of the polymer are the instrument of the imparting them the biological activity.

Keywords poly(urethane)s; biodegradation; human diseases, fungicidal; antimicrobial; action

1. Introduction

Under the blistering technical progress that was followed on the one hand by creation of new materials and from the other hand by the appearing of aggressive microorganisms’ population, the necessity of the control of this processes within new industrial environmental niche becomes more actual. Loss of industrial materials because of damage caused by biodeterioration, that was induced by microorganisms reach enormous sizes and make globally billions of dollars annually [1]. On the other hand a huge amount of industrial wastes that are mainly made up of synthetic polymers in recent decades has appeared. Theoretically it could be expected that synthetics will not be available for well-known destructors, because such compounds do not occur in nature and microorganisms that are adapted to them, are absent. However, the fact, that despite evident primitiveness organization, microorganisms, like all living systems, are characterized by anisotropic structure and are not insulated from external influences, was not taken into the consideration. In microorganisms it is exhibited by the level of specific enzymes that are responsible for activity and competitiveness even under the extreme conditions of reclamation of non-growing substrates [2]. Underestimation of these properties of microorganisms led to the fact that in recent decades appeared a huge amount of industrial wastes, that are mainly made up of synthetic polymers. Cesspool turned into natural sources of most active destructors, whose number of spores in the air dramatically increases. This phenomenon disquiets not only technical experts but also doctors, because these microorganisms (fungi) are causal factors of infectious-inflammatory diseases of humans and animals.

1.1 Systems and objects

1.1.1 Microorganisms (Fungi, Yeasts) – Polymers

The majority of the materials produced by man, including polymers, is susceptible to attack of environmental microorganisms that leads to their damage [3]. Influence of microorganisms on polymers is realized by two different processes: i) direct effect – is used for destruction and biodegradation of polymers, which is a native substance for microorganisms’ development; ii) indirect effect - is the influence of metabolic products of microorganisms. To evaluate the action of microorganisms on polymers exist several criteria: 1) biodeterioration - changing of chemical or physical properties of the material under the influence of microorganisms; 2) fungistatic effect – antimycotic influence on materials or antimicrobial treatment that protects the material from the fungi’s overgrowth in humidity conditions and 3) biodegradation [4]. Biodegradation [and biodeterioration] of polymeric material covers a great number of industry branches. Biodegradation of polymeric materials is a function of the polymeric materials structures, the condition of existence of microorganisms’ population that causes the biodeterioration of environmental conditions that contributes microorganisms’ development.

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Science against microbial pathogens: communicating current research and technological advances A. Méndez-Vilas (Ed.)______________________________________________________________________________

According to statistics, in which all kinds of induced by microorganisms biodegradation are included, it was determined that the major harm under conditions of environmental monitoring is caused by the fungi - micromicetes [5, 6]. More than 500 species of them that are considered to be active destructors of different polymeric materials are well-known. Biodegradation as a phenomenon is caused by the action of two dominant anthropogenic factors: increase of the rate of production and the adoption of deficient technologies and the intensification of technophilic species of fungi that contributes to the increment of infection load in the atmosphere as a result of the reclaiming of new territories. Under the influence of extreme environmental factors complexes of particularly aggressive species of microscopic fungi populations come into being. They have become ordinary components of industrial ecological niches that contaminate various materials, including well-known and also developed by modern technologies polymers. Processes of biodegradation of polymer materials are studied not sufficiently to control all their stages. It is indicated that not all micromicetes can be considered as active biodecomposers and specialized to a specific polymer material [7]. Mechanism of biodegradation action on polymers is also not determined and there are only general schemes, which envisage the process of destruction under the influence of individual microorganisms and their metabolites (enzymes, organic acids, etc.) and biological level of their adaptation possibilities. The mechanism of biodestruction includes a number of stages, which are carried out regardless of microorganisms species, as well as contributing factors and can be presented schematically as following [8]:

Specificity and complexity of the revelation of specialized micromycetes to the correlative polymer lies in the fact that it depends on the ecological valence of the strain rather than on species or genus. Most of these strains belong to the genus Aspergillis, Penicillium, Cladosporium, Fusarium. Their representatives are able to assimilate structural elements of the correlative polymers, which induce the biosynthesis of exoenzymes from oxidases’ and reductases’ groups, which actively affects the process of destruction. All kinds of microdestructors that were found on polymers display different activities during functioning indoors (in manufacturing enterprises and storehouses). Among all well-known contaminates (which occupy 360 species) 32 species were characterized as typical inhabitants of specific conditions. These species are Aspergillis and Penicillium. The are also well-know as contaminates of foodstuffs, different industrial materials and products, and on the assumption of their influence on human’s organism they are referred to the group of sorted out on the basis ”Bio Safety Levels”, potentially dangerous causative agents of opportunist humans’ illnesses [9, 10].

1.1.2. Microorganisms (Fungi, Yeasts) – Human

At present the processes of biodegradation, induced by micromycetes and by their metabolic products, including mycotoxins and endotoxins are studied in the medical and sanitary aspect as well. Toxins, which are produced my fungi, affect almost all human organs, especially if a person has problems with immune protection. Ecological environmental conditions have negative influence and it becoming worse (even in medical institutions) (Table 1 [11]). In addition, the human immune system worsens and it results in onset of mycosis, fungemia, allergies

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and opportunist diseases, causative agents of which are almost all species of micromycetes – the agents of biodegradation process. Fungi prevalence in life use is surprising. According to research data that was conducted in North America [12, 13] from 27 to 36 per cent of houses have problems with mold fungi. In Europe the mold fungi in houses was detected in Great Britain – from 17 to 46 per cent of houses, in the Netherlands – from 15 to 18 per cent of houses, in Finland – in 15 per cent of exploration. Penicillium (96%) і Cladosporium (89 per cent) were detected as often as not. In toto the authors [14] have found about 66 varieties of filamentous yeast fungus in 94 per cent of living quarters. Growth and colonization of microorganisms on the material surface can trigger an epidemiologic risky situation in the environment that is conditioned by large dissemination of microorganisms. It is especially evident, if a person stays in closed space, for example in cabins of space stations. During the operating of orbital station "Mir" it was stated an intensive conglomeration of microorganisms in the inner surfaces of hermetic cabin: bacteria and fungi [15]. Herewith, considerable influence during the biocenosis formation on the surfaces of hermetic cabin, have micromycetes, in particular fungi of species Aspergillis і Penicilliun. Their occurrence frequency in the conditions of orbital stations resembles the data received during the microbiological investigation of aeronauts from the spacecraft «Apollo» [16]. Microflora, especially fungi on the space board or on clothes and skin of its crew, becomes dangerous, if their amount increases and fungi change their biological characteristics [17]. Microbiological investigations carried out on the orbital station “Salut-6” displayed the connection between specific compound of environmental microflora and peculiarities of automicroflora conditions of upper air passages of cockpit personnel [18]. Such opportunistic species of fungi as Aspergillis niger, Aspergillis oryzae, Penicillium lanosum, can initiate toxic- allergic diseases when the immunity of a human organism is low. Besides, the micromycetes presented in station cabin can cause the environment air pollution with microadmixtures of toxic substances. Particularly the formation of ethanol and acetaldehyde is taking place during the development of mold fungi (Penicillium hryzogenum, Aspergillis niger, A. flavus, A. fumigatus) on the foam polyurethane’s surface [19]. Different species of micromycetes, which refer to Penicilliun, Aspergillis, Clostridium, Fusarium etc genus are detected in the orbital station “Mir” and are known as destructors. At the same time they can be the cause of great number of diseases, among them are aspergillosis, systemic mycosis, bronchopulmonit and also onychomycosis [20,21]. Thereby, all substrates contaminated be micromycetes, can be interpreted as sources of infection agents for environment [22].

Table 1 Specific environment spectrum: water (from water pipe, borehole), air and indoor dust in rooms of medical establishment.

Water Air Indoor dust in rooms

Aspergillis fumigatus Penicillium expansum Penicillium fellutanum Penicillium cyclopium Cladosporium cladosporioides Mycelia sterilia Mucor sp. Rhodotorula sp. Rhizopus oryzae Trichoderma linorum

Alternaria sp. Absidia corymbifera Aspergillis niger Aspergillis fumigatus Aspegillus terreus Aspergillis repens Aspergillis ochraceus Penicillium sp. Penicillium expansum Mucor spinosis Mucor plumbeus Mucor racemosus Gliocladium albicans Rhizopus oryzae Cephalocporium sp.

Penicillium Aspergillis Cladosporium Aureobasidium Acremonium Mucor Alternaria Trichoderma Candida Chaetomium Mortierella Rhizopus Ulocladium

A lot of scientific centers deal with the problem of polymer materials prevention from biodegradation action, inducted by microorganisms, and with the formation of polymer materials resistant to microdestructors’ action, and with the investigation of pathogenic action of fungi on human organism, and with the elaboration of the materials for preventive measures and mycosistreatment. However, it should be noted that these investigations are mainly aimed at development of materials or methods of prevention of either synthetics or people, and don’t deal with complex of problems for establishment of secure system of human and environment protection from fungi ravage. On the assumption of aforesaid, the aim of the investigation is the creation of biologically active polymer materials resistant to biodegradation, inducted by fungistatic/ fungicidal (antymycotic) microorganisms for human and environment protection.

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2. Metodology of the investigations

Principle concept of investigation is molecular design of polymer macromolecule by means of introduction of groups and fragments of relevant chemical structure using compounds [preparation] of fungicide [antimycotic] affect toward a large spectrum of biodestructors of polymer material which in the main time serve as etiologic factors for human diseases, and thus forming biologically active polymer material on this basis [8]. The main principles of creation of biologically active polymer materials. ● Biologically active compounds (BAC) interact with receptor by its active part [biophore] mostly due to deriving of weak non-valence bonds. An important role in such bonds is provided by Wan der Waals forces, hydrogen bonds, electrostatic interactions. Biophores’ structure may include heteroatoms (N, O, S, P), haloids, metals, heterocycles, groups –С=С–, and aroma structures in general. It is well known that certain low-molecular elementorganic compounds are effective mediators and considerably effective external factors for regulation of different biochemical processes, including lipogenesis in microorganisms. It is important to consider that the upper mentioned is determinative for adding biologic activity to monomer compounds, whereas for polymers one should consider also a topologic factor. From one side polymer matrix may facilitate availability of biophores, but from another it may block them. ● Design of macromolecule and its biophores due to introduction into the polymer chain structure of elements and fragments synergetic effect of which provodes the biologic activity. ●● Introducing BAC to the structure of macromolecule by means of chemical bonding ●● Structural modification of polyurethanes allows regulating their supramolecular organization, which serves as an instrument of variation both a place of localization of biophore and its availability. ● Task-oriented choice of modifiers. ●●Using of modifying additives apt to complex formation with polyurethane macrochain fragments, which leads to their strong fixation of polymer matrix and impedes a diffusion of these compounds on the polymer surface with their further removal. ●● Use of biologically active natural compounds apt to weak physical fixation in polymer matrix as modifying additives. Such polymers are characterized not only by contact [fungistatic/fungicidal] effect, but also by controlled extraction of BAC into the environment. The upper mentioned plays a great role in guaranteed protection of human located for a long time in closed habitat. ● Mainly due to the complex of specific physic-chemical properties and opportunity of task-oriented structural-chemical, physical and phase modification, polyurethanes constitute the most attractive class among many polymers for using as matrices for producing of biologically active polymer materials.

3. Biologically active polymers: Structure-properties relationships

3.1 Polyurethanes modified with inorganic and metal-containing additives.

Polyurethanes (PUs) of the general structure: [-R2-OCHN-R-NHCOO-R1-OCONH-R-NНCO-]n-, where R – residuum of 4,4’-diphenylmethane, R1 – remainder of oligotetramethyleneglycole (ММ 1000), R2 – remainder of dihydrazides isophtalic acid; modified with inorganic salts ZnCl2, SnCl2 and transition metal acetyl acetonates, Мt(АсАс)n - a) Zn(II), Cu(II), Pb(II) and b) Sn(IV)dichloride [23]:

O CCH

CMt

O

OCCH

C O

CH3

CH3

CH3

CH3 a) b) Colonizing polymer materials fungi produce specific ferments (apt to splitting any chemical bonds) and organic acids (tartaric, citric, lactic, oxalic, malic acid, etc) facilitating of acid hydrolyze of substrates [11]. Physic-chemical properties of certain PUs, modified by metal-containing additives are described in Table 2. The level of PUs’ bipodeterioration was estimated by means of deformation-strength retention coefficients: Кδ= δ1/δ ·100% и Кε=ε1/ε·100%, where δ, ε и δ1, ε1- tensile strength and elastic elongation of film materilas “before” and “after ” influence of microorganisms: Aspergillis oryzae, Aspergillis niger van Tieghem, Aspergillis terreus Thom, Chaetomium varioti Bainier, Penicillium funiculosum Thom, Penicillium chrysogenum Thom, Penicillium cyclopium, Trichoderma viride Pers. ex Fr according to [4, 24].

C O

Sn

CH3

CH

C O

CH3

CO

CH

C

CH3

O

CH3

Cl

Cl

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Table 2 . Physic-mechanical and biological properties of PUs.

Properties of initial PU Properties after the biological tests (PU′) Samples , МPа

rel.,% , МPа

rel.,% Degree of

biodeterioration, % Fungistatic activity after20/30 days, (points) Кσ Кε

PU-1 76,4 350,0 72,5 358,9 94,9 102,5 4 PU-1M1 66,2 337,5 60,0 250,0 90,6 94,1 1/0 PU-2 54,8 400,0 41,0 566,6 74,9 141,6 5/5 PU-2M2 47,1 525,0 40,36 558,3 95,7 106,4 1/1

Biodeterioration level – (alternation of physic-mechanic indicies) of modified PU is insignificant, which is proved by keeping the К and Кε coefficients on high level: К =90,6-95,7 %, Кε =94,1-106,4 %. Investigations of biological activity PU have shown that PU-1М1 and PU-2М2, containing modifying additives: ZnCl2, SnCl2 and Zn(AcAc)2 (4 % wt.) and ZnCl2 and Zn(AcAc)2 (2,5 % wt.), accordingly, possess fungicide properties which are estimated by 0-1 points [7], whereas their matrixes PU-1(based on dihydrazide of isophtalic acid) and PU-2 (based on 1,4-butane diol) are not fungicides: their influence on microorganisms is estimated by 4-5 points [25]. The influence of microorganisms on polyurethane microheterogeneous structure was studied by means of small angle X-ray scattering (SAXS) method. Analysis of performed (Fig.1a SAXS profiles) modified PU-1M1 (Fig.1, curve 1) and affected by micromicetes PU′-1М1 (Fig.1, curve 2) testifies the effect of microorganisms on the polymer structure. The scattering profile of initial PU-1М1 (composition+) is characterized by poorly expressed maximum of intensity which indicates structural heterogeneity – existence of periodicity in spatial location of hard and soft domains in the bulk of initial PU. Period of alternation (D) of heterogeneity microzones of the same electronic density comes to [26] around 13,4 nm. Micromicetes influence (Fig. 1, curve 2) leads to disappearance of interference maximum on scattering profile and simultaneously to enhancing of scattering intensity (curve 2), which as is well known occurs while increasing of size of microareas of heterogeneity in the polymer bulk. Semiqauntative estimation of the structure heterogeneity level of studied PU, estimated by comparison of the values of Porod invariant (Q) [26] ,shows that affected by micromicetes PU (PU′-1М1) is characterized by significant decrease of heterogeneity level in polymer volume (304 and 861 relative unit, respectively) while the sizes of hard domains are increased. These alternations prove the participation of microorganisms’ metabolism products in structuring processes in polymer volume. The role of such structuring agents may be performed by metabolism products (organic acids, eg. citric acid, producer of mold fungi of Aspergillis genus, promoting the formation of specific intermolecular interaction, including H-bonds between macrochains and metabolites.

a) b)

Fig. 1 а) SAXS profiles: initial PU-1М1(1) and affected by micromicetes PU′-1М1 (2); б) SAXS profiles: initial PU-2 (1), PU-2М2 (2) and affected by micromicetes PU′-2 (1′) and PU′-2М2 (2′ ). The comparison of SAXS profiles of patterns of PU-matrix PU-2 (1) of composition 1 (Fig. 1b) and PU-2М2 (2) of composition 2 before and after they were affected by micromycetes has shown that the character of influence of micromycetes on PU-2М2 differs from those for PU′-1М1. The influence of micromycetes did not lead to visible changes in microgeneous structure of PU-2М2 (Fig. 1b, curves 1,1′ and 2, 2′), that was proved by the values of Porod invariant (Q) PU-2М2 and PU′-2М2 - 465 и 455 relative unit, correspondingly. SAXS data are confirmed by IR-spectroscopic research. As the result of influence of micromycetes on PU-1М1 the intensity of absorption band of bonded semicarbazide carbonyls (1670 sm-1) increases in comparison with similar band of free urethane carbonyls (1730 sm-1). There is no an evident change of absorption band 1540 sm -1 - δ(NH) free as the result of influence of micromycetes. A slight intensity reduction of the band δ(NH) free - 1540 sm -1 for PU′-2 confirms the reduction of quantity of free NH-groups, which confirms X-ray data re structurize influence of metabolites. Analysis of IR-spectra of PU-2 - PU-2М2 , PU′-2 - PU′-2М2 and multiple ATR IR spectra taken from the surface of films testify to slight

0 ,6 1 ,2

8 0

1 6 0

I , о т н . е д .

q , н м -1

2

1

0 , 6 1 , 2

1 2

2 4

3 6

2 '

1 '

2

1

I , о т н . е д .

q , н м- 1

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reduction of intensity of band δ(NH)free- 1540 sm -1 and increase of intensity of band 1705 sm-1 comparatively to band 1730 sm -1. This indicates the changes in hard domains’ order owing to H-bond association of urethane carbonyls and micromycetes’ metabolism products which correlates with X-ray data. Coordinating centers generated by complex-formation of modifiers with PUs hard blocks’ polar groups may serve as additional microareas of heterogeneity in given PUs. Their structure can be performed as [25]:

NHCO

O

NH C O

OZn

2+

Generation of such coordinating centers is confirmed by DSC and WAXS [27] data. Thus, the stable to biodetereoration PU-materials possessing the fungicidal activity in relation to the mold fungi - the most aggressive microorganisms-biodestructors as well as the causal factors of human disease - have been obtained by PU modification with Zn and Sn compounds.

3.2. Polyurethanes, comprising macroheterocyclic fragments in the backbone

PU of general structure: [-R2-OCHN-R-NHCOO-R1-OCONH-R-NНCO-]n- , where: R2 –dihydrazide disulfodibenzo -18-crown-6 (c, d) residue[28]: c) d) Biological activity research of synthesized PUs showed (Fig 2), that PU-4(based on d) possesses fungicide activity of 0 points. It totally suppresses the growth of mold fungi, polymer surface remains clear. PU-3(based on c) possesses fungistatic activity of 1 point – the growth of molt fungi is appreciably inhibited; there are insignificant colonies on its surface. All polymers are stable to biodegradation.

Fig 2 Bioactivity of PU: PU-1(1), PU-2(2), PU-3(3), PU-4(4). 1 point conforms to 10% on axis ordinate.

They save their physic-mechanical characteristics even after been influenced by fungi. The values of К and К reach 95 - 100%, while the values of К and К for PU-1 (based on dihydrazide diphenyl metane disulphoacid) are 4550 % [28]; at that the polymer does not posess fungicide activity. Macrochain structure of PU-1, PU-3, PU-4 differs only by presence or absence of crown ether(CE) fragments and all other elements and groups which may be a part of biophores composition heteroatoms (N, O and S, aromatic structures, sulfonylhydrazide groups) are present in the structure of all studied PUs. Based on this we can conclude that level of biological activity of synthesized PUs basically depends on CE fragments built in microchain, due to their capacity to specific and mainly electrostatic interaction with biological objects. Although the presence in PUs structure of CE fragments is necessary but not sufficient factor of biological activity indication. As PU-2 based on diamine dibenzo-18-crown-6, differs from PU-3(c) and PU-4(d) only by the absence of sulfonyl amide group, and characterized by the total absence of biological activity – 5 points. It can be as the result of different access of biophores to reseptor because of differences in supermolecular PUs structure. Thus according to the data of Multiple ATR IR spectra taken from the surface of PUs, the consentration of CO-groups bonded with sulfonyl groups and CE fragments on PUs surface is maximal for PU-3 and PU-4 but minimal for PU-2 [28], i.e. the biophores’ accsess and its activity for these polymers are differrent. So, the presence of CE fragments in

SO2

NHNH2

H2NHNO

2S O

O

O

O

O

O

H2NHNO

2S SO

2NHNHO

2S

O

O

O

OO

O

O

O

O

OO

O SO2NHNH

2

N N C

OHH

N N C

OHH

ZnO

O

O

O

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macrochain is necessary but not sufficient factor defined their activity. An aditional factor is the character of topology of polymer patterns, biophores access, which depends on supramolecular structure of PUs.

3.3 Poly(аmide)urethanes

Poly(amide)urethanes (PAU) of general structure:[-R2-OCHN-R-NHCOO-R1-OCONH-R-NНCO-]n-, where: R2 – residue of Мt(АсАс)n: Zn(II), Cu(II), Pb(II) and Sn(IV)dichloride, correspondingly, (a) and (b) [29]. Analysis of biodeterioration level of PAU/Мt testifies to the high level of maintaining of their physic-mechanical properties under the influence of mold fungi: Кδ=0,99-0,96, and Кε =0,89-0,87. PAU/Sn, PAU/Zn (Fig. 3) and PAU/Cu show contact fungicide (0-1 point) effect, PAU/Pb – fungistatic effect (2 points), showing also an inhibit effect on the processes of spore germination, growing processes and spore reproduction of micromicetes test-culture even at non-direct contact with fungi spores. There were no germinating or turgescent spores found on the surface of studied PAUs. Viability preserving of spores on

0

2

4

6

8

10

days

samples1 2 3

Fig. 3 Inhibiting of the process Fig. 4 Micromicetes spores vitality on the

of spore PAU/Zn germination. PAU surface: 1- PAU/Cu, 2-PAU/Zn, 3- PAU/Sn.

the PUs surface was studied by imprint method on nutrient medium. It was determined that spore viability is kept on PAU/Sn during three days, on PAU/Zn – during six days, and on PAU/Cu – during ten days (Fig. 4). It was shown that on the expiry of these periods the spores were lack of vitality. One should notice that among 38 standard cultures of bacteria, yeastlike and micellar fungi, the fungistatic effect on PAU/Cu up to ten days was occurred for Aspergillis niger and Penicillium cyclopium; on PAU/Zn – up to 6 days for Aspergillis terreus, and on PAU/Sn – up to 3 days for Penicillium cyclopium. Other types of spores which were used for contamination were not found [30].

Fig. 5 Multiple ATR IR spectra taken from the film surface of PAU/Сu(1), PU-2/Cu(acAc)2 (2) and PU-2 (3).

IR- data allow both to prove the structure of obtained materials and to venture an assumption as to the reason of their biologic activity. The research has shown that (Fig.5 ATR IR spectra) relative intensity of the amide fragments 1640см-

1 is considerably higher than intensity of urethane fragments. This may testify to the presence on the PAU surface layer

500 1000 1500 3000 3500 4000

1550 1600 1650 1700 1750 1800

3

2

1

, сm -1

1658

1705

1730

1640

Re

fle

cti

on

3

2

1

, сm-1

1658

1705

1730

1640

Re

flect

ion

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of a greater fraction of polymer chains parts containing the Мt(АсАс)n fragments in comparison with polymer volume. Herein, the relative intensity of the bond 1640см-1 of PAU/Sn exceeds the same for PAU/Cu that indicates the greater concentration of -R2-OCHN- fragments, thus the same concerns Sn(IV) atoms in the surface layers of PAU. Such peculiarity of polymer surfaces may explain both general high PAU activity regarding microorganisms and also the greater activity of PAU/Sn.

3.4 Metalcarboxylated polyurethanes

Metalcarboxylated polyurethanes (MPU) of general structure [31]: [-R2-OCHN-R-NHCOO-R1-OCONH-R-NНCO-]n-, where R2:

O

OO

OH OH

OO

OMt2+

e) MPU possess activity towards mold fungi. Their boideterioration level is: Кσ = 92,5% and Кε = 88,6% whereas for PU of similar structure on the basis of 1,4 –butane diol (R2) it is 50 and 49%, respectively. Steam, moisture permeability and density of MPU-films do not change in the result of microbiologic influence. MPU are fungicides towards fungi: Aspergillis oryzae, Aspergillis niger van Tieghem, Aspergillis terreus Thom, Chaetomium varioti Bainier, Penicillium funiculosum Thom, Penicillium chrysogenum Thom. Introduction of covalent-bonded metal monohydroxypentylphthalates (R2) leads to formation

Pseudo-phase, enriched with Cu-containing fragments

Fig. 6 Optical microscopy data of MPU/Cu. of two pseudo-phases with educing of phthalate fragments containing –COO-+Mt+-OOC– grouping into a separate phase (Fig. 6). In accordance with multiple ATR IR spectra data the concentration of such grouping on the surface of MPU is higher than in volume [32], which may determine the biologic activity of MPU.

3.5 “End-capped” modified polyurethanes

3.5.1 PU of linear structure

PU with biologically active compounds as terminal groups (from 20% to 40% mass.) in the structure, have been obtained by blocking the terminal isocyanate groups of PU with those biologically active compounds. The structure of PU may be performed as [33]: [-R2-OCNH-R–NHCOO-R1-OCONH–R-NНCO-]n–R3, where: R2 – dihydrazide of isophthalic acid residue, R3 - 5-nitro-8-oxyquinoline hydrochloride residue NОQ-HC, (f) and sulfonylhydrazide of diphenyl(2-chlorphenyl)-1-imidazolylmethane DCIМ-SH, (g):

N

NO2

OH ClH f) g) PU possess both antibacterial (Table 3.), and fungicide activity of prolonged effect (Fig.7).

NN

Cl

SO2NHNH2

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Table 3 Antibacterial properties of PUs.

Test-culture NОQ-HC DCIМ-SH E.coli 8026 15 0 E.coli ACC25922 20 sd.gr 23 sd.gr E.coli 055 20 0 E.coli 6724 18 18 E.coli 6775 15 0 Kl.pneumoniae 50/55 16 20 Kl.pneumoniae 6467 15 0 Kl.pneumoniae 8061 12 0

S.aureus 6652 23 25 S.aureus АТСС 25923 18 8 S.epidermidis 43 15 20 Pr. mirabilis F-403 18 sd.gr 25 sd.gr Pr. mirabilis 7096 25 0

sd.gr – secondary growth

1а) 1б) 2)

Fig.7 Fungicidal activity of “еnd-capped ” modified PU: PUS/ NОQ-HC (40%): 1а – primary, 1б – repeated contamination;

2 – PU/ NОQ-HC. Fungicide effect that was also accompanied by strong inhibiting influence on spore reproduction, pigmentation and change of color has been remained.

3.5.2 Polyurethanes of branched structure

PU produced by means of transformation of terminal hydrazide group of 1,1-dymethylhyrazine [34] based PU to the structures of hydrazinium type [33]:

OCHN R NCOO R 1 OCONH R NCO NH

N+

CH3

CH3R 2NHCO R HNOCO R 1 OOCHN R HNOC

[

[

]n

[ ] Cl

,

where: R2 – residues of sulfonylchlorides of 5-nitro-8-oxyquinoline hydrochloride (NОQ-SH) (h) and of diphenyl(2-chlorphenyl)-1-imidazolylmethane DCIМ-SH(i):

h) i)

Microbiological research of the surfaces of PU/ NОQ-SH and NОQ-SH showed the lack of the growth of the following yeasts: С. albicans 5335, С. albicans 6539, C. tropicalis, C. tropicalis 5819, C. parapsilosis 4599, C. parapsilosis 890, C. guilliermondii 6811, C. guilliermondii 6811, C. guilliermondii 412, C. krusei 79. Microheterogenity structure of such PU is determined by the presence of terminal group situated at hard - soft domains interface. This group serves as biologically active fragment of macromolecule, that allows to vary the place of biophore location and its availability for receptor.

NOH

NO2

SO2Cl

NN

Cl

SO2Cl

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3.6. Polyurethane foams

3.6.1 Elastic polyurethane foams

PUF of general structure: -[CE-DIC-PE-DIC]n-, CLA -[CE-DIC-PE-DIC]n-

where: PE – bi- and three functional ether and/or ester residues (MM 800-5000), DIC – 2,4(2,6)-tolyelene diisicyanate resedue, CE – bifunctional chain extender residue, CLA – cross-linking agent (diisocyanate/macrodiisocyanate) [35]. Biologically active natural compounds (BAC) were used for physic modification of PU: manuca oil, eucalyptus oil, lavender, grapefruit oil as well as synthetic macromolecule compound diphenyl (2-chlorophenyl)-1-imidazolylmethane (k) [36]: k) PUFs, modified with natural BAC (Fig. 8) display fungistasis towards Aspergillis niger, Aspergillis oryzae, Aspergillis terreus, Chaetomium globosum, Penicillium brev-compactum, Penicillium chrysogenum, Penicillium cyclopium, Penicillium funiculosum, Penicillium martensii, Penicilliumochrochloron, Paecilomyces varioti, Fusarium moniliforme, Trichoderma viride, Alternaria alternate by blocking the process of conidie formation and increasing the term of pores’ germination and colonies formation (lag-phase) in 4-8 times in comparison with control (1).

Fig. 8 Duration of lag-phases (term of pores’ germination and colonies formation) of different types of micromycetes on PUF/BAC compounds: 1- PUF; 2-5 - PUF/ BAC, where BAC vegetable oils: 2- eucalyptus; 3 - lavender; 4 - manuca; 5 – grapefruit.

PUF, containing 5-nitro-8-oxyqinoline, montmorillonite and manuca oil possess double effect: both antimycotic (Table 4) and bacteriostatic (Table 5).

Table 4 Fungicidal (antimycotic) properties of PUF. Table 5 Antimicrobial properties of PUF.

Yeast-like fungi Diameter of the growth retardation zone, mm

С. albicans 3220 6-15 С. albicans 5335 9-12

С. albicans 5717 9-14 С. albicans 6539 10-15 C. tropicalis 6-15 C. tropicalis 5819 6-17

C. parapsilosis 890 6-12 C. guilliermondii 6811 6-14 C. krusei 791 8-16

Bacteria cultures Diameter of the growth retardation zone, mm

E.coli 6332 11-13 E.coli 8026 10-14 E.coli АСС 25922 9-13 K. pneumoniae 1739 8-14

P. aeruginosa 8062 4-8 P. vulgaris 8718 10-14 P. mirabilis 6500 8-12

P. mirabilis 7037 10-14 S. aureus 6878 6-15

Fungicidal (antimycotic) of PUF has prolong nature which retains not less than two years (Fig. 9)

NN

Cl

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1) 2)

Fig. 9 Fungicidal activity of PUF: 1- PUF/MMT/manuka oil; 2 – PUF/DCIM (after 2 year storage ).

3.6.2 Two-component foam polyurethane material

Unlike the PUF (according # 3.6.1) two-component PUF/2 is formed directly on the protected surface, small period of its forming (5-6 minutes) is provided by using isocyanate component (DIC) – the mixture of prepolymer (TDI/Bifunctional oligipropylene glycol) and TDI. PUF contains the following biocides (diphenyl (2-chlorophenyl)-1-imidazolylmethane (k), 1,10 -decamethylene-bis (N, N – dimethyl- metoxycarbonilmethyl) - ammonium dichloride (l) and manuca oil (MO) [37]: l) Fungistasis of PUF/2 was proved in relation to mold fungi according to [4, 24]. The growth of Aspergillis oryzae, Aspergillis niger van Tieghem, Aspergillis terreus Thom, Chaetomium varioti Bainier, Penicillium funiculosum Thom, Penicillium chrysogenum Thom, Penicillium cyclopium, Trichoderma viride Pers. ex Fr under conditions of direct contact of the lower surface of patterns with infected environment both on and under PUF patterns is absent. Patterns PUF/2 possess also antimicrobial activity against bacteria - E. coli АТС 25922, Е. со1і 150, Klebsiella pneumoniae 6447, S. aureus 180, Pseudomonas аeruginosa 561, Pseudomonas аeruginosa 6847, Proteus mirabilis F-403, Proteus mirabilis 6054, Proteus vulgaris 8718 and yeasts - Candida albikans ATCC 10231, Candida parappsilossis 890, Candida krusei 791, Candida tropicalis, Saccharomyce cerev 4888 completely suppressing their growth. Carried out experiment with bacteria has shown the growth retardation zone around the samples for the following genus: E. Coli, Klebsiella and Staphylococcus – 10-15 cm, Pseudomonas – 9-11 cm and Proteus – 10-22 cm. Toxic investigations of PUF/2 were accomplished by way of microbiological control of their toxicity during formation directly on the surface of surgical stitch of animal being operated. For that mixed components were placed on nutrient agar (ХН 222 Himedia) which contained 5% of human erythrocytes [0(1)Rh + group]. Patterns were aged for 72 hours at the temperature of 370 С, whereupon - again for another 72 hours at the temperature of 220 С. With the aim of finding out potential toxicity of these patterns in the process of “ageing” PUF/2, the same patterns after 14 days were placed on fresh blood agar and were keeping under observation according to the above mentioned procedure. Toxicity tests of obtained PUF has shown that all these PUF patterns do not evince toxicity in relation to erythrocytes, and do not result in their hemolysis, including the period after PUF “ageing”.

3.7 Toxicological study of obtained PU

The unique criterion of chance to introduce the elaborated PU-materials into practice is conducting of toxicological research, including: i) sanitary-chemical and ii) toxic-hygienic estimation of the material. From the entire list of obtained PU-materials, the following were subjected to tests: PU-2M2; PU-2/Cu(acAc)2; PAU/Cu, PAU/Sn, PUF(c). Sanitary-chemical tests were carried out according to [38]. PUs samples without direct contact with human body were examined with the help of sanitary chemistry methods and by express method of tissue culture. The indices of migration of organic and reduction impurities into the simulative environment are essentially lower in comparison with admissible hygienic standards (table 6).

CH3

CH

CH3H3C

OCOCH2 N

CH3

CH3

(CH2)10N

CH3

CH3

CH2OCO

CH3

CH3

CH

CH3

_+ +2CI

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Table 6 Content of reducing agent impurities and change of pH in daily extracts (n=3).

Sample Reducing agent impurities Change of рН PU-2M2 0 +0,30 PU-2/Cu(acAc)2 0,6 +0,55 PAU/Sn 0,6 +0,80 PAU/Cu 0,9 +0,05 PUF(c) 0 +0,40

Obtained results testify to the sufficient stability of PUs and correlate with the histotoxicity indices (HI), determined by the method of tissue culture on white outbreed rates [38]: at the level (0.78-0.74) 0.01 at the norm for non-toxic polymer degree HI 0.72. The sample PU-2M2was subjected to the complete toxic-hygienic tests using rates and rabbits, and it was determined the follows: allergenic and irritating effect of polyurethane extracts; - toxic influence of polyurethane extracts on animals under the experiment; - pathomorphological studies of innards; - biochemical ration of blood; - general reactivity of organism, functions of central nervous and cardiovascular systems, kidneys, liver, protein metabolism. Toxic-hygienic studies of PU-2M2testify to th absence of its toxic influence.

4. Conclusions

Structural-chemical and physical modification of polyurethane allows to introduce into the macromolecule structure of biologically active compounds as chain extenders and/or terminal groups and/or incorporate them into the polymeric matrix by including into the macromolecule structure by covalent bonds formation, complex formation, formation of ionic and ion-hydrogen bonds with the macromolecule groups and units. The control of polymers’ supramolecular structure enable to concentrate on their surface layer the groups/units of biophores: -R2(Mt)-OCHN-, –COO-+Mt+-OOC–, crown ether sulfonyl derivatives, increasing thereby an accessibility of biophores. The level of polymers biodeterioration is determined by their supramolecular structure, degree of crystallinity and microphase segregation. Including of metal containing fragments into the polyurethane macromolecule structure or their rigid fixation by means of complex forming with PUs macrochain fragments enable to avoid a diffusion of metal containing component onto the polymer material surface, that exclude their toxicity towards the living organisms. Thus, materials with fungal/fungistatic activity towards a large spectrum of biodestractors have been developed; materials stable to induced by microorganisms corrosion, which have anti-microbial activity and are able to inhibit the spores’ growth of pathogenic microorganisms on their surface and, thus, to exclude the causative factors of infectious-inflammatory human and animal diseases have been obtained.

Acknowledgments The author gratefully acknowledges the assistance of Prof. E.Koval, Prof. A.Rudenko, Dr.V.Veselov and colleagues from the Department of Chemistry of Heterochain Polymers & Interpenetrating Polymer Network.

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polymer materials against the microorganism's attack

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