Alternative Synthesis Route of Biocompatible Polyvinylpyrrolidone

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Alternative Synthesis Route of Biocompatible Polyvinylpyrrolidone Nanoparticles and Their Effect on Pathogenic Microorganisms Vedran Milosavljevic, Pavlina Jelinkova, Ana Maria Jimenez Jimenez, Amitava Moulick, Yazan Haddad, Hana Buchtelova, Sona Krizkova, Zbynek Heger, Lukáš Kalina, Lukas Richtera, Pavel Kopel, and Vojtech Adam Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.6b00807 • Publication Date (Web): 12 Dec 2016 Downloaded from http://pubs.acs.org on December 15, 2016

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Molecular Pharmaceutics

Alternative Synthesis Route of Biocompatible Polyvinylpyrrolidone Nanoparticles and Their Effect on Pathogenic Microorganisms Vedran MILOSAVLJEVIC1, Pavlina JELINKOVA1, Ana Maria JIMENEZ JIMENEZ1,2, Amitava MOULICK1,2, Yazan HADDAD1,2, Hana BUCHTELOVA1, Sona KRIZKOVA1,2, Zbynek HEGER1,2, Lukas KALINA3, Lukas RICHTERA1,2, Pavel KOPEL1,2 and Vojtech ADAM1,2* 1

Department of Chemistry and Biochemistry, Mendel University in Brno, Zemedelska 1, CZ-

613 00 Brno, Czech Republic 2

Central European Institute of Technology, Brno University of Technology, Technicka

3058/10, CZ-616 00 Brno, Czech Republic 3

Materials Research Centre, Faculty of Chemistry, Brno University of Technology,

Purkynova 118, Brno 612 00, Czech Republic

*Correspondence: [email protected]; Tel.: +420-545-133-350; Fax: +420-545-212044

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Abstract Herein we describe a novel alternative synthesis route of polyvinylpyrrolidone nanoparticles using salting-out method at a temperature close to polyvinylpyrrolidone decomposition. At elevated temperatures, the stability of polyvinylpyrrolidone decreases and the opening of pyrrolidone ring fractions occurs. This leads to cross-linking process, where separate units of polyvinylpyrrolidone interact among themselves and rearranges to form nanoparticles. The formation/stability of these nanoparticles was confirmed by transmission electron microscopy, X-ray photoelectron spectroscopy, mass spectrometry, infrared spectroscopy and spectrophotometry. The obtained nanoparticles possess exceptional biocompatibility. No toxicity and genotoxicity was found in normal human prostate epithelium cells (PNT1A) together

with

their

high

haemocompatibility.

The

antimicrobial

effects

of

polyvinylpyrrolidone nanoparticles were tested on bacterial strains isolated from the wounds of patients suffering from hard-to-heal infections. Molecular analysis (qPCR) confirmed that the treatment can induced the regulation of stress-related survival genes. Our results strongly suggest that the polyvinylpyrrolidone nanoparticles have great potential to be developed into a novel antibacterial compound.

Keywords: Antibacterial; Cross-linking; Polyvinylpyrrolidone nanoparticles; Salting-out; Toxicity


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Introduction The use of nanomaterials, especially nanoparticles, in human medicine can provide universal tools for drug delivery, sensing, bio-imaging, tissue engineering and development of therapeutic agents. The importance of nanoparticles is reflected in their properties such as solubility, diffusivity, blood circulation half-life, drug release characteristics, and immunogenicity. Application of nanoparticles as therapeutic and diagnostic tools for medical purposes is getting more attention in the recent years, especially for treatment of cancer, asthma, diabetes and bacterial infection.1 However, potential toxicity of nanoparticles still remains one of the main concerns among scientific reports, as it is mostly limited by presence of heavy metals.2 To overcome the problem of nanoparticles toxicity, the applications of biomaterials with high biodegradability and biocompatibility are highly recommended. Polymers, one of biomaterials, are able to diminish the toxic effects and provide numerous biological applications in therapy and diagnostic.3 Polymers represent materials with unique properties due to their favorable chemical stability, biocompatibility, and low toxicity in comparison with other materials. Polymers offer many advantages, especially in nanoparticle production for medical purposes, such as site specific drug delivery, controlled release of drugs as long circulating drug carriers and antimicrobial activity in combination with various metals.4 The easy way and cost effectiveness of the synthesis of the polymer nanoparticles can also be considered as benefits of these nanoparticles; however their applications especially in medicine is still under discussion.5 Currently, various types of methods are reported for the synthesis of polymeric nanoparticles e.g. rapid expansion of a supercritical solution such as solvent evaporation,6 salting-out,7 nanoprecipitation,8 supercritical fluid technology,9 or polymerization techniques, such as micro-emulsion, mini-emulsion and surfactant-free emulsion.10



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Production of antimicrobial nanoparticle requires surface modification that can minimize the impact of surface-defect, trap sites, and direct quenching from the surrounding.11 Such a functionalization with polymers ensures the covering of surface with carboxyl or hydroxyl groups that can specifically interact with biological molecules and act as antibacterial agent, opening the door for using these particles as the fastest route to take commercial medical application.12 As mentioned before, various known techniques can be utilized in order to produce polymer based nanoparticles. However, the synthesis of polymeric nanoparticles by standard methods results in relatively large nanoparticles which limits the crossing of the nanoparticles through different biological barriers or causes poor drug release in case of a drug delivery.13 To eliminate these problems, it is highly required to use proper method with control of stirring speed, temperature and polymer concentration.14 Based on these considerations we were interested in designing and synthesis of polyvinylpyrrolidone nanoparticle (denoted as PVP-NPs) which will not be limited by their large size. We wanted to avoid the use of the starting precursors and surface modifications by exploring the best synthetic route, which will be more economical and utilizing greener chemistry. Polyvinylpyrrolidone (PVP) was selected as a polymer source due to its biocompatibility, as it will later stand a better chance in applications requiring low toxicity tolerance. Due to the limited size options we designed an alternative synthesis route combining saltingout and thermal decomposition of PVP to produce particles of lower size and increased bioavailability. It is well known that thermal decomposition (oxidative degradation) starts around 230°C.15 Due to that, the thermal decomposition of PVP was conducted in ethylene glycol at 230°C in presence of potassium hydroxide as a salting-out agent to prevent the mixing of PVP with ethylene glycol. At elevated temperature, the stability of PVP decreases


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and leads to open the pyrrolidone ring fractions. This phenomenon results in the transfer of hydrogen atoms from one radical to another, which leads to the formation of double bonds by hydrogen donor and saturation of acceptor by cross-linking process. Due to the cross-linking process induced by heating, separate units of PVP interact between themselves forming PVPNPs.16 PVP-NPs were tested for antimicrobial activity against Gram positive and Gram negative bacteria. Molecular analysis on Escherichia coli was used to understand the model of action of PVP-NPs and expression of genes involved in oxidative stress. Our designated PVP-NPs show exceptional biocompatibility in eukaryotic cells. Results and Discussion Characterization of PVP-NPs The characterization steps were carried out on dried samples after removing all impurities from the solution and lyophilization. The morphology of PVP-NPs was investigated by transmission electron microscopy (TEM). PVP-NPs have spherical shape and a representative single particle size around 10 nm as shown in Figure 1A. The TEM images show that the most of the particles formed chain-like aggregates, which mostly arise from the draining of samples before the measurements. Despite that, the TEM pictures evidenced that spherical particles are formed. To confirm the results obtained from TEM, dynamic light scattering (DLS) was applied for characterization of size and size distribution (Figure 1B). The average sizes of PVP-NPs were found to be 9±2 nm which is in agreement with the results from TEM (Figure 1A). The size comparison of PVP-NPs with the size of PVP molecules (2±1 nm) confirmed nanoparticles formation. To obtain the further insight into the PVP-NPs nature, the measurements of zeta potential were conducted. It was shown that the PVP-NPs have potential at -29.9 mV (Supplementary figure S1A); indicating good colloidal stability. The PVP molecules showed zeta potential at -15.5 (Supplementary figure S1B). Additionally, we confirmed that the elevation of temperature has strong correlation to decrease in particle size. 5 ACS Paragon Plus Environment


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The results of DLS and scanning electron microscope (SEM) for the PVP-NPs synthesized by conventional salting-out method in acetone (boiling point 56°C) revealed that the average size of the nanoparticles is around 141±20 nm (supplementary Figure S2A and S2B). This procedure formed an oil-in-water emulsion which led to acetone diffusion in aqueous phase and results in production of large nanospheres.17 In our previous work we have reported that polymers can provide surface functionalization of carbon nanoparticles and this directly effects to increase the fluorescence properties.18 To elucidate the optical properties of PVP-NPs, spectrophotometric characterization was performed. The absorbance maximum of PVP-NPs was at 280 nm (Supplementary Figure S3A). Thus, λex of 280 nm was exploited to determine the λem of PVP-NPs. The obtained results showed that λem of PVP and PVP-NPs were 460 nm or 420 nm, respectively. It is obvious from Figure 1C that instead of the PVP-NPs, PVP exhibited only negligible fluorescence. This corresponds to our previous statement that the PVP-NPs have great fluorescence properties which come from polymer functional moieties, as it was also reported by Kuehne et al.19 The fluorescence of the PVP-NPs did not change after two months, proving its exceptional stability (Supplementary figure S3B). The chemical composition of the PVP-NPs surface was tested by X-ray photoelectron spectroscopy (XPS). The XPS of C 1s spectrum ascribed four components to achieve an acceptable fit (Figure 1D). The main peak with the binding energy at 285.0 eV is attributed to the adventitious carbon creating the polymer chain (–C–C–). Another two components represent two different kinds of bond between carbon and nitrogen. The first one at 285.4 eV belongs to the carbon in five membered ring (N5) of the polymeric N heterocyclic compound (–CH2–N–), while the second one at 286.3 eV is typical for the carbon in polymer chain with –CH–N– connection. Finally, the peak at 288.0 eV indicates the –C=O double bond in N– heterocyclic system. There is also a strong K 2p doublet in the C 1s spectrum which is


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presented due to the addition of KOH during the PVP-NPs synthesis. The major component in N 1s spectrum at 399.8 eV (Figure 1E) is assigned to the nitrogen in pyrrolidone system. This shows that under thermal decomposition, the chemical structure of PVP was not significantly altered. This was also proven by the results obtained from elemental analysis (CHNS analysis) (Supplementary Table S1) and energy-dispersive X-ray spectroscopy (EDX), confirming a uniform distribution of the elements in PVP-NPs. EDX proved that carbon, oxygen and nitrogen in the morphology of examined area are equally distributed (and Supplementary Figure S5).

Figure 1: The characterization of PVP-NPs. (A) TEM images of PVP-NPs in magnifications at 100 kx and 200kx. (B) Size distribution of PVP-NPs and PVP. (C) Emission maxima of PVP-NPs and PVP measured within the range from 230–850 nm using the λex 280 nm. (D) Curve fitted high resolution XPS C 1s spectrum. XPS operated at 150 W (10 mA, 15 kV) using an analysis area of ~300 × 700 µm. (E) Curve fitted high resolution XPS N 1s spectrum. For confirmation and better understanding of the chemical composition of the PVP-NPs, Fourier transform infrared spectroscopy (FTIR) measurements were conducted (Figure 2A). In the spectrum of PVP-NPs there are only slight differences noticeable in comparison to PVP. The most intense signal is in the fingerprint area, also called Amide II region, and there is a signal at 1550 cm-1 which belongs to the PVP-NPs. The presence of weak band in this 7 ACS Paragon Plus Environment


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region can be explained as the opening of pyrrolidone ring and formation of (-R1-C(O)-NHR2) amidic group. It can also not be excluded that the presence of the peak in this region is a result of double bond formation of oxygen to nitrogen atom or/and scissoring of NH2 group. However, it is clear that the presence of the peak at 1550 cm-1 belongs to oxidative degradation of pyrrolidone ring during thermal decomposition.20 The peak at 1047 cm-1 presented in the spectra of PVP-NPs can be assigned to C-O vibration, which is also connected with oxidation process. Furthermore, the PVP-NPs were studied by matrix-assisted laser desorption/ionization timeof-flight mass spectrometry (MALDI-TOF MS), which revealed that the molecular weight distribution of PVP-NPs is shifted to lower mass region of 3-5 kDa (Figure 2B). This can be attributed to high polydispersity of PVP which directly influences molecular weight distribution shifting to lower values in MALDI-TOF-MS analysis.21 However, it is clear that the mass of our particles contains repeating units of PVP-111 Da.


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Figure 2: (A) FTIR spectra of PVP-NPs and PVP recorded by spectrometer equipped with platinum-attenuated total reflectance accessory with diamond crystal. (B) MALDI-TOF spectra of PVP-NPs using following matrices: α-cyan-4-hydroxycinnamic acid and sinapinic acid.

Evaluation of biocompatibility of PVP-NPs The biocompatibility of PVP-NPs was investigated on PNT1A and LNCaP cell lines by estimating the growth rate inhibition after 24 hours using following concentrations of PVPNPs: 0; 0.2; 0.4; 0.8; 1.6; 3.1; 6.2; 12.5 and 25 µg/mL. As shown in Figure 3A, PVP-NPs did not induce any toxicity in nonmalignant PNT1A cells. Testing on LNCaP malignant cells revealed 30% decrease in viability in the highest concentration (Figure 3B). Recent study on HeLa and Jurkat cells suggests internalization of nanoparticles by endocytosis due to high hydrophilicity of polymeric nanoparticles and negatively charged carboxylic group.22 The activity of the polymeric nanoparticles is mostly connected with conjugated/encapsulated drugs or by the surface modifications using various biologically active molecules. It is a known fact that the toxicity of the polymer particles in cancer cells comes from the risk of a “lysosomal storage disease” syndrome, which is connected with high dose of polymer in cells.2 Haemolytic assay was further carried out to study the haemocompatibility of the PVP-NPs. The haemolytic activity of the nanoparticles is a crucial parameter to estimate their therapeutic index. Strong membrane transfer ability of the PVP-NPs to cells could also threaten RBCs via membrane-disruption mechanisms. Figure 3C illustrates that the highest applied concentration of the PVP-NPs (16 µg/mL) triggered only negligible haemolysis (about 7.4% related to 100% caused by Triton X-100 as positive control). Thus, it can be noted that the PVP-NPs can be applicable with minimal effect on blood circulation.



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Figure 3: Cytotoxicity tests of designated PVP-NPs assayed using the (A) benign epithelial prostate cells (PNT1A) and (B) malignant prostate cells (LNCaP). Cytotoxicity was analyzed using the MTT assay. (C) Haemocompatibility of PVP-NPs tested with human RBCs. PBS (pH 7.4) was utilized as negative and Triton-X-100, which causes 100% hemolysis, as positive control. Comet assay analyses single- and double strand breaks of DNA caused by the carcinogens. The intensity of the comet tail relative to the head reflects the number of DNA breaks in cells. Figure 4A illustrates that only negligible genotoxicity was found within the tested concentration range of the PVP-NPs, when compared to positive control (H2O2) which induces strands breakage and loss of supercoiled structure. Manual scoring (Index of damage, which show the influence of treatment on DNA fragmentation) of 50 randomly selected comets revealed that higher DNA damage was caused after 2 h exposure, however the extent of damage was small enough to be repair by the cells reparation apparatus (observable as apparent decrease of Index of damage in 24 h treatment in Figure 5B). Similarly, the damage level expressed as Comet grade revealed that the highest possible damage (grade 4) was found only in case of H2O2 treatment. Comet grade is additional information representing the


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distribution of distinct types of comets within a treated sample. The classification is made based on the shape and the fluorescence of the comet tail [0 (no visible tail) to 4 (significant DNA in tail)]. Taken together, our results demonstrate that our designed PVP-NPs are highly biocompatible with eukaryotic organisms.

Figure 4: Comet assay for DNA damage. (A) Fluorescence microscope imaging of PNT1A cells treated with different concentrations of PVP-NPs (6; 12 and 24 µg/mL) for 2 h (upper) and 24 h (lower). DNA was stained by PI, scale bars: 500 µm. (B) Index of damage and (C) Comet grade compared to negative control (PBS, pH 7.4) and positive control (150 µM H2O2). All data are represented as average ± standard deviation (n = 3). Antibacterial effect of PVP-NPs The multidrug resistances of the microorganisms have become a clinical and public health problem.23 Application of polymers in prevention of bacterial infection is still limited on surface modification of metal nanoparticles.24 Our work describes novel PVP-NPs, which exhibit good effect in the antimicrobial activity in Gram negative and Gram positive bacteria, as was evidenced using 5 Gram negative bacteria (E. coli, P. mirabilis, P. aeruginosa, K. pneumoniae and E. cloacae) and 4 Gram positive bacteria (S. aureus, S. pyogenes, E. faecium and E. faecalis). To determine the antimicrobial activity of the PVP-NPs we analyzed the 11 ACS Paragon Plus Environment


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growth curves after the application of PVP-NPs during 24 hours. The bacterial growth after the application of PVP was also analyzed. IC50 (represents inhibitory concentration to 50% growth) was also calculated. It was found that PVP does not possess any antimicrobial effects (Figure 5A and 5B). In the figure 5A (testing on Gram negative bacteria), a significant decreasing impact on bacterial growth was observed after the exposure of the PVP-NPs. In the opposite case, for P. aeruginosa, an increased growth after the treatment with 4 µg/mL (p = 0.048) and 8 µg/mL (p = 0.129) of PVP-NPs was observed. A significant decrease of the growth curve in E. coli at concentrations of 4 µg/mL (p = 0.066) and 8 µg/mL (p = 0.030), in P. mirabilis at 4 µg/mL (p = 0.035), in K. pneumoniae at concentration of 2 µg/mL (p = 0.034) and 4 µg/mL (p = 0.014), in E. cloacae at 8 µg/mL (p = 0.009) was detected. In the figure 5B for Gram positive bacteria, a general decrease of the growth in comparison to the control group was observed after an incremental addition of PVP-NPs, however only significant decreases for S. pyogenes, E. faecium and E. faecalis were observed at concentrations of 2 µg/mL and 4 µg/mL. No significant decrease change was found in S. aureus for all different concentrations of PVP-NPs. Half maximal inhibitory concentration confirmed our results, the IC50 values for the bacteria are as follows: E. coli 5.2 µg/mL, P. mirabilis 6.1 µg/mL, P. aeruginosa 6.2 µg/mL, K. pneumoniae 4.1 µg/mL, E. cloacae 5.6, S. pyogenes 6.3 µg/mL, E. faecium 4.7 µg/mL, and E. faecalis 5.6 µg/mL (Figure 5C). In case of S. aureus IC50 showed higher concentration of applied concentration (13.2 µg/mL). Our results suggest a potent antibacterial activity of PVP-NPs when compared to the polymeric nanomaterial synthesized by Carmona-Ribeiro et al.25 The explanation is likely to be that the alternative way of synthesis resulted in small nanoparticles ( 5 ' exonuclease activities in Escherichia coli endonuclease IV: Structural and genetic evidences. Mutat. Res.-Fundam. Mol. Mech. Mutagen. 2010, 685, (1-2), 70-79. 34. Bhat, A. P.; Shin, M.; Choy, H. E., Identification of High-Specificity H-NS Binding Site in LEE5 Promoter of Enteropathogenic Esherichia coli (EPEC). J. Microbiol. 2014, 52, (7), 626-629. 35. Krin, E.; Danchin, A.; Soutourina, O., Decrypting the H-NS-dependent regulatory cascade of acid stress resistance in Escherichia coli. BMC microbiol. 2010, 10. 36. Stella, S.; Falconi, M.; Lammi, M.; Gualerzi, C. O.; Pon, C. L., Environmental control of the in vivo oligomerization of nucleoid protein H-NS. J. Mol. Biol. 2006, 355, (2), 169174. 37. Nagarajavel, V.; Madhusudan, S.; Dole, S.; Rahmouni, A. R.; Schnetz, K., Repression by binding of H-NS within the transcription unit. J. Bio. Chem. 2007, 282, (32), 2362223630. 38. Grainger, D. C.; Goldberg, M. D.; Lee, D. J.; Busby, S. J. W., Selective repression by Fis and H-NS at the Escherichia coli dps promoter. Mol. Microbiol. 2008, 68, (6), 1366-1377. 39. Vijayakumar, S. R. V.; Kirchhof, M. G.; Patten, C. L.; Schellhorn, H. E., RpoSregulated genes of Escherichia coli identified by random lacZ fusion mutagenesis. J. Bacteriol. 2004, 186, (24), 8499-8507. 40. Sandoval, J. M.; Arenas, F. A.; Vasquez, C. C., Glucose-6-Phosphate Dehydrogenase Protects Escherichia coli from Tellurite-Mediated Oxidative Stress. PLoS One 2011, 6, (9). 41. Prozorov, A. A., The bacterial cell cycle: DNA replication, nucleoid segregation, and cell division. Microbiol. 2005, 74, (4), 375-387. 42. Elnakady, Y. A.; Chatterjee, I.; Bischoff, M.; Rohde, M.; Josten, M.; Sahl, H. G.; Herrmann, M.; Muller, R., Investigations to the Antibacterial Mechanism of Action of Kendomycin. PloS One 2016, 11, (1). 43. Zhou, K.; Zhou, L.; Lim, Q. E.; Zou, R.; Stephanopoulos, G.; Too, H.-P., Novel reference genes for quantifying transcriptional responses of Escherichia coli to protein overexpression by quantitative PCR. BMC Mol. Biol. 2011, 12. 44. Peng, S.; Stephan, R.; Hummerjohann, J.; Tasara, T., Evaluation of three reference genes of Escherichia coli for mRNA expression level normalization in view of salt and organic acid stress exposure in food. FEMS Microbiol. Lett. 2014, 355, (1), 78-82. 45. Berney, M.; Hammes, F.; Bosshard, F.; Weilenmann, H. U.; Egli, T., Assessment and interpretation of bacterial viability by using the LIVE/DEAD BacLight kit in combination with flow cytometry. Appl. Environ. Microbiol. 2007, 73, (10), 3283-3290. 46. Evans, B. C.; Nelson, C. E.; Yu, S. S.; Beavers, K. R.; Kim, A. J.; Li, H.; Nelson, H. M.; Giorgio, T. D.; Duvall, C. L., Ex Vivo Red Blood Cell Hemolysis Assay for the Evaluation of pH-responsive Endosomolytic Agents for Cytosolic Delivery of Biomacromolecular Drugs. J. Vis. Exp. 2013, (73), 1-5. 47. Wasfi, R.; Elkhatib, W. F.; Khairalla, A. S., Effects of Selected Egyptian Honeys on the Cellular Ultrastructure and the Gene Expression Profile of Escherichia coli. PLoS One 2016, 11, (3). 48. Lu, Z.; Li, C. M.; Bao, H.; Qiao, Y.; Toh, Y.; Yang, X., Mechanism of antimicrobial activity of CdTe quantum dots. Langmuir 2008, 24, (10), 5445-5452. 35 ACS Paragon Plus Environment


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49. Wu, H.; Wang, H.; Chen, J.; Chen, G.-Q., Effects of cascaded vgb promoters on poly(hydroxybutyrate) (PHB) synthesis by recombinant Escherichia coli grown microaerobically. Applied Microbiology and Biotechnology 2014, 98, (24), 10013-10021. 50. Wong, M. L.; Medrano, J. F., Real-time PCR for mRNA quantitation. Biotechniques 2005, 39, (1), 75-85. 51. Livak, K. J.; Schmittgen, T. D., Analysis of relative gene expression data using realtime quantitative PCR and the 2(T)(-Delta Delta C) method. Methods 2001, 25, (4), 402-408.


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The characterization of PVP-NPs. (A) TEM images of PVP-NPs in magnifications at 100 kx and 200kx. (B) Size distribution of PVP-NPs and PVP. (C) Emission maxima of PVP-NPs and PVP measured within the range from 230–850 nm using the λex 280 nm. (D) Curve fitted high resolution XPS C 1s spectrum. XPS operated at 150 W (10 mA, 15 kV) using an analysis area of ~300 × 700 µm. (E) Curve fitted high resolution XPS N 1s spectrum. 96x52mm (300 x 300 DPI)

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Figure 2: (A) FTIR spectra of PVP-NPs and PVP recorded by spectrometer equipped with platinum-attenuated total reflectance accessory with diamond crystal. (B) MALDI-TOF spectra of PVP-NPs using following matrices: α-cyan-4-hydroxycinnamic acid and sinapinic acid. 82x60mm (300 x 300 DPI)



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Cytotoxicity tests of designated PVP-NPs assayed using the (A) benign epithelial prostate cells (PNT1A) and (B) malignant prostate cells (LNCaP). Cytotoxicity was analyzed using the MTT assay. (C) Haemocompatibility of PVP-NPs tested with human RBCs. PBS (pH 7.4) was utilized as negative and TritonX-100, which causes 100% hemolysis, as positive control. 154x103mm (220 x 220 DPI)


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Comet assay for DNA damage. (A) Fluorescence microscope imaging of PNT1A cells treated with different concentrations of PVP-NPs (6; 12 and 24 µg/mL) for 2 h (upper) and 24 h (lower). DNA was stained by PI, scale bars: 500 µm. (B) Index of damage and (C) Comet grade compared to negative control (PBS, pH 7.4) and positive control (150 µM H2O2). All data are represented as average ± standard deviation (n = 3). 80x49mm (300 x 300 DPI)



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Determination of bacteria growth after the PVP-NPs treatment. Student’s paired t test was used to compare mean difference change in growth between E. coli before and after treatment with different concentrations of PVP-NPs and PVP. (A) Percentage of Gram negative bacterial growth after application of various concentrations of PVP-NPs using method of growth curves after 24 h of measurement. (B) Percentage of Gram positive bacterial growth after application of various concentrations of PVP-NPs using method of growth curves after 24 h of measurement. (C) IC50 calculations of PVP-NPs for Gram negative and Gram positive bacterial strains. 83x60mm (300 x 300 DPI)


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Effects of PVP-NPs (0; 2; 4 and 8 µg/mL) on E. coli assayed by Colony forming test; under optical microscope in ambient light and microscopic assay for evaluation of live/dead treated with PI/SYTO9. 96x67mm (300 x 300 DPI)



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qPCR on E. coli elucidating the gene response of treated bacteria. (A) Initial gene expression involved in transcription control and cell division. (B) Expression of gens after 1; 2 and 5 h treatment by PVP-NPs. (C) RNA quality evaluates by bleach gel method. (D) Reference gene to total RNA. (E) Total RNA yield after 0; 1; 2.5 and 5 h. (F) Mechanism of action of response to PVP-NPs stress. 158x153mm (220 x 220 DPI)


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Alternative Synthesis Route of Biocompatible Polyvinylpyrrolidone

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