Glucose-modified carbosilane dendrimers: Interaction with model membranes and human serum albumin

Journal Pre-proofs Glucose-modified carbosilane dendrimers: interaction with model membranes and human serum albumin Dominika Wrobel, Monika Müllerová, Tomá š Straš ák, Květoslav Rů žička, Michal Fulem, Radka Kubíková, Maria Bryszewska, Barbara KlajnertMaculewicz, Jan Malý PII: DOI: Reference:

S0378-5173(20)30122-8 https://doi.org/10.1016/j.ijpharm.2020.119138 IJP 119138

To appear in:

International Journal of Pharmaceutics

Received Date: Revised Date: Accepted Date:

13 August 2019 7 February 2020 11 February 2020

Please cite this article as: D. Wrobel, M. Müllerová, T. Straš ák, K. Rů žička, M. Fulem, R. Kubíková, M. Bryszewska, B. Klajnert-Maculewicz, J. Malý, Glucose-modified carbosilane dendrimers: interaction with model membranes and human serum albumin, International Journal of Pharmaceutics (2020), doi: https://doi.org/ 10.1016/j.ijpharm.2020.119138

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Title:

Glucose-modified carbosilane dendrimers: interaction with model membranes and human serum albumin Authors: Dominika Wrobel1, Monika Müllerová1,2, Tomáš Strašák1,2, Květoslav Růžička3, Michal Fulem3, Radka Kubíková1, Maria Bryszewska4, Barbara Klajnert-Maculewicz4 and Jan Malý1

Affiliations: 1

Faculty of Science, Jan Evangelista Purkinje University, Usti nad Labem, Czech Republic

2

Institute of Chemical Process Fundamentals of the CAS, v.v.i, Prague, Czech Republic

3

Department of Physical Chemistry, University of Chemistry and Technology, Prague, Technická 5, CZ-166 28 Prague 6, Czech Republic

4

Department of Biophysics of Environmental Pollution, Faculty of Biology and Environmental Protection, University of Lodz, Lodz, Poland

Corresponding author: Dominika Wrobel Jan Evangelista Purkyne University Department of Biology České mládeže 8 400 96 Ústí nad Labem Czech Republic tel: +420 475 283 622 fax: +420 475 283 622 e-mail: [email protected]

Keywords: Glucose-modified carbosilane dendrimers; liposomes; model lipid membranes; membrane fluidity; differential scanning calorimetry; circular dichroism; proteins

1

Abstract Glycodendrimers are a novel group of dendrimers (DDMs) characterized by surface modifications with various types of glycosides. It has been shown previously that such modifications significantly decrease the cytotoxicity of DDMs. Here, we present an investigation of glucosemodified carbosilane DDMs (first–third-generation, DDM1-3Glu) interactions with two models of biological structures: lipid membranes (liposomes) and serum protein (human serum albumin, HSA). The changes in lipid membrane fluidity with increasing concentration of DDMs was monitored by spectrofluorimetry and calorimetry methods. The influence of glycodendrimers on serum protein was investigated by monitoring changes in protein fluorescence intensity (fluorescence quenching) and as protein secondary structure alterations by circular dichroism spectrometry. Generally, all generations of DDMGlu induced a decrease of membrane fluidity and interacted weakly with HSA. Interestingly, in contrast to other dendritic type polymers, the extent of the DDM interaction with both biological models was not related to DDM generation. The most significant interaction with protein was shown in the case of DDM2Glu, whereas DDM1Glu induced the highest number of changes in membrane fluidity. In conclusion, our results suggest that the flexibility of a DDM molecule, as well as its typical structure (hydrophobic interior and hydrophilic surface) along with the formation of larger aggregates of DDM 2-3Glu, significantly affect the type and extent of interaction with biological structures.

1. Introduction Cancer is a significant health problem and is one of the ten main leading medical causes of death (Ferlay et al., 2018). The most frequently used method for cancer treatment is chemotherapy, but this approach has several limitations due to the high risk of side effects that are harmful for the patients. The limitation of anticancer therapies based on chemotherapeutic agent administration to the bloodstream is associated with low cellular uptake into the cancer cells and several undesirable toxicity effects, such as nephrotoxicity, hepatotoxicity and cardiotoxicity (Devita et al., n.d.; Rang et al., 2012). This negative impact of anticancer drugs on patients stimulated the investigations of new administration methods, which will decrease side effects and increase drug bioactivity. One of the strategies is to use nano-scale carriers for selective cancer cell drug targeting. Due to the size, shape, stiffness and functional groups present on their surface, nanoparticles penetrate cells via different pathways. DDMs are characterized by having good biocompatibility and flexibility of structure, both of which can help to transform them into more selective platforms for drug delivery. Generally, the idea of polymer conjugates or complexes with a drug is based on the 2

motivation of improved solubility of the drug in water and prolongation of drug circulation in the bloodstream. To increase drug delivery efficiency, various strategies have been investigated, and several types of nanoparticles have been applied. There are many examples of nanoparticles used as drug delivery platforms for anti-cancer therapies. These systems comprise a large range of materials including lipids (liposomes), polymers (micelles, DDMs, hyperbranched polymers, dendrons), viruses or organometallic compounds (nanotubes) (Anselmo and Mitragotri, 2014; Guo et al., 2020; Jinadatta et al., 2017; Sherje et al., 2018; ud Din et al., 2017). There are several commercially available anti-cancer medicaments based on nanoparticles. Zinostatin, one of the oldest anti-cancer drugs to be based on a polymer-protein conjugate targeting the hepatocellular carcinoma, was FDA approved in 1993. Another example of polymer-drug conjugate pharmaceutics is Prothecan, which is based on a poly(ethylene glycol) conjugate with camptothecin and is used for small-cell lung cancer treatment. Marqibo, a drug delivery system consisting of vincristine sulphate encapsulated in liposomes and used for treating acute lymphoblastic leukaemia, was approved in 2012. Recently, nanodrugs based on drug-antibody conjugates for the treatment of various malignant cells have been approved; for example, Adcentris and Perjeta (Jain et al., 2014). Among the several types of drug delivery systems, the DDMs represent a class of nanomaterials with high potential for improving the therapeutic effect of anticancer drugs (Farooq et al., 2019). DDMs are promising nanoparticles as drug delivery platforms in anticancer therapy due to their well-defined structure and flexibility. Carbosilane DDMs (CS-DDMs), which represent types of silane-based hyperbranched polymers, have been investigated since the early 1990s (Jadvar, 2016). Due to their hydrophobic interior and hydrophilic surface, they were considered as carriers for hydrophobic molecules in otherwise polar environments (Zhou and Roovers, 1993). In the context of polymer therapeutics, carbosilane DDMs have been studied for many purposes, as have been, for example, drug/nucleic acids (small interfering RNA, anti-sense oligonucleotides etc.) delivery systems in anti-cancer/anti-HIV therapy as well as a biologically active nanomaterials themselves (e.g. due to antiviral and antimicrobial properties) (PedziwiatrWerbicka et al., 2012; Raviña et al., 2010; Weber et al., 2008). Carcinogenesis is a multistep process during which several cell-imposed barriers are eliminated (Weinberg, 2013). This process results in the evolution of so-called cancer cells, which demonstrate changes in their metabolic programs, including increased uptake of glucose, enhanced rates of glutaminolysis and fatty acids synthesis (Weisburger, 1989). Changes in cell metabolism further support tumour cell growth and survival. Many cancer cells overexpress

3

glucose transporter 1 (GLUT1), an important member of the glucose transporter protein (GLUT) family and which transports D-glucose across cell membranes (Niu et al., 2014). In normal tissue, GLUT is present in large numbers of isoforms. Fourteen members of the mammalian facilitative glucose transporters (GLUT1-GLUT12, GLUT14 and HMIT) have been identified. All are intrinsic membrane proteins differing in tissue-specific expression and are metabolically and hormonally regulated. Every type of glucose transporter is characterized by different affinities for glucose and other hexoses. One of the common GLUT transporters is GLUT1 transporter, which is widely distributed in normal tissues and is expressed at high levels in endothelial and epithelial-like barriers of the brain, eye, peripheral nerve, placenta and lactating mammary gland (Zhao and Keating, 2007). Interestingly, a relation between overexpression of the GLUT1 transporter and the carcinogenesis process has been shown. Overexpression of this transporter has been reported in many cancers including breast, pancreatic, brain, lung, renal, oesophageal, cutaneous, hepatic, colorectal, ovarian, endometrial and cervical (Szablewski, 2013; Zhao and Keating, 2007). Based on the abnormal behaviour of cancer cell lines and GLUT1 overexpression, new strategies of anticancer treatment were recently outlined based on glucose-anticancer drug conjugates targeting GLUT1 (Jadvar, 2016). As an example, enhanced anti-hepatocarcinoma efficacy due to GLUT1 targeting has been observed by the use of PAMAM DDMs conjugated to glucose and anti-cancer drug camptothecin (Ma et al., 2018). Inspired by these promising approaches, we have recently synthesized and tested for their in vitro and in vivo toxicity new types of first–third-generation carbosilane glycodendrimers (DDM1-3Glu) (Liegertová et al., 2018). Although our investigations revealed a relatively large disproportion between the toxic influence on animal cells and dechorionated embryos of the model fish Danio rerio, the overall cytotoxicity was low—indicating that DDM1-3Glu is a promising candidate for their use in drug delivery applications. The combination of a hydrophobic interior and the presence of hydrophilic glucose units on the periphery of DDM1-3Glu offer the possibility of low water-soluble anticancer drug encapsulation and its transport into cancer cells by active targeting on the GLUT1 receptor with the advantage of relatively low cytotoxicity and side effects. In addition to the properties and interactions of anti-cancer drug nano-carriers with the targeted and non-targeted cells, drug administration is also associated with other factors that can influence drug delivery efficiency. To get to the targeted place and to be active inside the cell, the nano-carrier/drug complex must overcome many challenges during its transportation in the bloodstream. Interaction of nano-carriers with the serum proteins could degrade them or 4

effectively prevent them from penetrating through the cell membrane. The most abundant protein in plasma is human serum albumin (HSA), which is known for its highly efficient ligand binding capacity. This property of HSA makes it an important factor in the investigation of the pharmacokinetic behaviour of drug nano-carriers (Vilanova et al., 2016). The formation of protein corona or nanoparticle corona (based on mutual size of nanoparticles and proteins) (Shcharbin et al., 2015) can significantly influence the final fate of the nano-carrier in the bloodstream, toxicity, biodistribution, the efficiency of its action and excretion (Wrobel et al., 2017). It has been shown that the strength and type of DDM-plasma protein interactions are related to DDM generation and the functional groups present on their surface (Gabellieri et al., 2006; Jokiel et al., 2006). It has also been demonstrated that DDMs with peripheral cationic functional groups are more likely to interact with albumins as compared to other types of DDMs (Klajnert et al., 2003). Despite this, investigations of neutral (hydroxyl group-terminated) DDM interactions with albumins have illustrated that they can interact with proteins through hydrogen bond formation (Klajnert et al., 2008; Wrobel et al., 2017). Therefore, interaction studies of glucose-modified carbosilane DDMs with serum proteins is another important step in the investigation of their potential use as drugdelivery nano-carriers. Similarly, cell membranes themselves influence the distribution and transport of internalized nanoparticles within cells. Nano-carriers, which must be delivered inside the cell successfully, have to overcome the cell membrane and/or must be able to escape from endosomes after their cellular uptake via endocytosis. Nanoparticles that interact with the cell membranes can also significantly change their properties. It has been confirmed that one of the possible toxic effects of nanoparticles can be correlated with the influence of DDMs on cell membrane properties and integrity (Hong et al., 2004; Ionov et al., 2012; Klajnert et al., 2006; Mecke et al., 2005, 2004; Wrobel et al., 2015b). Experiments focused on the monitoring of nanoparticle interaction with model lipid membranes can therefore suggest the mechanisms of their toxicity and reveal an eventual further negative influence on membrane properties. There has been relatively little investigation so far dedicated to interaction studies of sugar-modified hyperbranched polymers and model biological systems such as proteins or lipid membranes. Two types of sugar-modified hyperbranched polymers, poly(propylene imine) (PPI) and poly(ethyleneimine) (PEI) have been most frequently studied (Appelhans et al., 2009; Ciepluch et al., 2012; Ciolkowski et al., 2012b; Franiak-Pietryga et al., 2013; Janaszewska et al., 2012; Klajnert et al., 2008; Wrobel et al., 2017, 2015a, 2015c; Ziemba et al., 2011). In both the lipidic and protein model systems, the conclusion was drawn that the changes in their structures were related to hydrogen bonding between polymer and the model system investigated 5

(Ciolkowski et al., 2012b; Klajnert et al., 2008). Previous experiments suggested that despite some of these glyconanoparticles possessing a positive charge on their backbone, the electrostatic interactions of the charged groups did not play a major role in the interaction process. Here, we continue this line of research with the analysis of DDM-protein and -lipid membrane interactions with a recently introduced novel type of first–third-generation glucose-modified carbosilane DDM. The presented work is another logical step in the research of drug-delivery application potential of this new type of DDM nanoparticle.

2. Materials and methods

2.1

Materials

Lipids:1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and fluorescent probes: 1,6diphenyl-1,3,5-hexatriene phenylammonium

(DPH);

p-toluenesulphonate

N,N,N-trimethyl-4-(6-phenyl-1,3,5-hexatrien-1-yl) (TMA-DPH);

4-(2-hydroxyethyl)piperazine-1-

ethanesulfonic acid (HEPES buffer) were purchased from Sigma Chemical Company. Three generations of glucose-modified carbosilane DDMs (with -D-glucopyranosyl units as functional groups) (Fig.1) were synthesized and characterized according to published protocols (Liegertová et al., 2018). All DDMs were dissolved in 10 mM HEPES with 0.2 mM EDTA buffer pH 7.4.

2.2

Liposomes preparation

Neutral and pure DMPC liposomes were used. To prepare liposomes, phospholipids were dissolved in an organic solvent—chloroform. Appropriate amounts of lipid solution with or without fluorescent probes were placed in a flask under a stream of nitrogen to evaporate the solvent. The final lipid concentration was 500 μM, and the phospholipid/fluorescent probe molar ratio was 500:1. The obtained lipid film was hydrated with an appropriate volume of 10 mM HEPES with 0.2 mM EDTA buffer pH 7.4 and mixed. The lipid suspension was incubated at 37 °C, which is the temperature above the lipid phase-transition point (the transition temperature of DMPC is 24 °C). Subsequently, the lipid-buffer mixture was forced to pass >15 times through a polycarbonate membrane of 100 nm porosity (Nuclepore, T-E) mounted in a mini-extruder (Avanti Polar Lipids) fitted with two 1000-μl Hamilton gastight syringes. A polycarbonate membrane of 100 nm porosity was used for liposome formulation due to the results of our previous test with larger 400 nm porosity membranes. Liposomes obtained with larger 400 nm porosity membranes were characterized by higher size distribution in comparison to those 6

formulated on a 100 nm porosity membrane. The suspension of liposomes with a better-defined size was chosen as the bilayer model. After the liposome formation procedure, the suspension of liposomes was incubated in a water bath at 37 °C for 10 minutes. 2.3

Fluorescence anisotropy measurements

Fluorescence anisotropy measurements were carried out with a FluoroMax-4 spectrofluorimeter (Horiba Scientific, France). Two fluorescent probes were used in order to monitor membrane fluidity in a whole bilayer. The first, an apolar molecule—DPH—was incorporated into the hydrophobic region of the liposome bilayer, and the second, with a hydrophilic group—TMADPH—was anchored on the surface of the liposome bilayer and exposed to a hydrophilic environment due to its positively charged amino groups. The excitation and emission wavelengths were 340 nm and 428 nm, respectively. The slit width of the excitation monochromator was 3.0 nm for both labels, whereas the slit width of the emission monochromator was 5.0 nm for DPH and 5.5 nm for TMA-DPH (Hirsch-Lerner D. and Barenholz, 1998; Shinitzky and Barenholz, 1978, 1974). During all experiments, a temperature of 37 °C was stably controlled. The final lipid concentration in the sample was 100 μM, and the phospholipid/fluorescent probe molar ratio was 500:1 (Shinitzky and Barenholz, 1978, 1974; Wrobel et al., 2012). The anisotropy values (r) of the samples were calculated by the fluorescence data manager program (FluorEssence software) using the following equation: r = (IVV - GIVH)/(IVV + 2GIVH) where IVV and IVH are the vertical and horizontal fluorescence intensities, respectively, to the vertical polarization of the excitation light beam. The factor G = IHV/IHH (grating factor) corrects the wavelength response to the polarization of the emission optics and detectors. 2.4

Differential Scanning Calorimetry

The differential scanning calorimetry (DSC) method was used to analyse DDM interactions with neutral DMPC liposomes. A pure solution of liposomes or liposome/DDM-buffered suspension samples were loaded into the measurement cell of a Tian-Calvet type micro-calorimeter (SETARAM µDSCIIIa, France). As a reference, an empty cell was used during measurements. The typical lipid concentration was 7.4 mM, and a 50:1 lipid:DDM molar ratio was used. Three heating/cooling cycles at a range of 5–45 °C were performed at a 0.5 °C/min scan rate (Wrobel et al., 2018, 2015a, 2011).

2.5

Fluorescence quenching of HSA 7

Fluorescence spectra of HSA were measured in increasing concentrations of glucose-modified carbosilane DDMs. In each sample, the final concentration of HSA was 10 M, and the concentration of glucose-modified carbosilane DDMs was in the range of 5 M to 200 M (DDM:protein molar ratios of 1:2; 1:1; 2:1; 6:1; 10:1; 15:1; 20:1). The excitation wavelength was 295 nm, and the excitation slit used during measurements was set to 3 nm. The emission slit was 4.5 nm, and the spectra were recorded from 310 nm to 500 nm. From corresponding HSA quenching spectra, spectra obtained for each DDM concentration were extracted. The binding constants were calculated from the modified Stern-Volmer equation: F0/F=1/(faKa[Q])+1/fa where fa is the fraction of the fluorescence that is accessible to the quencher, Ka is the SternVolmer quenching constant of the accessible fraction and [Q] is the concentration of quencher (Lakowicz, 2006).

2.6

Circular dichroism (CD) measurements

CD spectra were measured with a Jasco J-815 Spectrometer (Japan). The concentrations of HSA for all measurements was 1M, and the protein solution was prepared in 0.5 mM PBS at pH 7.4. Samples were placed in a cuvette of 0.5 cm path length (Böhm et al., 1992). Concentrations of glucose-modified carbosilane DDMs were altered from 0.5 to 20 M (DDM:protein molar ratios of 1:2; 1:1; 2:1; 6:1; 10:1; 15:1; 20:1). The spectrometer was continuously purged with dry nitrogen before and during the experiment. The spectra were recorded from 195 to 260 nm. Spectral data were given as the mean residue ellipticity. The secondary structure composition of the proteins was examined using CD deconvolution with K2D3 web server (Perez-Iratxeta and Andrade-Navarro, 2007).

2.5

Statistical analysis

Statistical analysis and exponential curve fitting were performed using GraphPad Software. The distribution was determined with a Student's t-test. Results are expressed as the mean  the standard error of the mean (S.E.M.) or standard deviation (S.D.).

8

3. Results

3.1

Fluorescence anisotropy measurements

Fluorescence anisotropy experiments were run at physiological temperature (37 °C), which is a temperature above the main transition points of both lipids. At set experimental conditions, all types of experimental bilayers possessed a disordered structure (liquid crystal phase). The changes in anisotropy value in the presence of the DDM1-3Glu are shown in Fig. 2. All investigated DDMs generally interacted with neutral DMPC, influenced lipid bilayers and changed their properties. Interactions between liposomes and DDMGlu were related to concentration and DDM generations. The highest number of changes in membrane fluidity were caused by the first-generation DDM—DDM1Glu—and its overall influence was different from that of the other two generations. DDM1Glu induced the destabilization of the lipid bilayer, even at low concentrations, as demonstrated by a reduced anisotropy value. In contrast, when the concentration of DDM1Glu was further increased from its initial values, a simultaneous increase of anisotropy was observed. This type of interaction of DDM1Glu with lipidic membranes was observed with both fluorescence probes used. The strength of DDM-lipid bilayer interaction was also correlated with the DDM generation. The most significant increase of anisotropy (stronger interaction) was induced by DDM1Glu, whereas second- and third-generation DDMs (DDM12Glu)

showed lower anisotropy changes (weaker interaction). Moreover, first- and third-

generation DDMGlu interacted more strongly with the hydrophobic part (larger changes in anisotropy of DPH probe) than with the hydrophilic part of the lipid bilayer (lower changes in anisotropy of TMA-DPH probe). The interactions of DDM2Glu with both parts of the lipid bilayer were comparable.

3.2

Differential Scanning Calorimetry

Increasing the temperature of pure lipids changed their molecular organization from the gel phase at low temperature to the liquid-crystalline phase at a higher temperature. The temperature at which the structure changes its property is referred to as the transition point. Above this temperature, molecules move more easily and quickly and possess higher energy. In the case of liposomes, the so-called pre-transition peak is not usually detected, and only the main transition is visible in the thermograms (Fig. 3). DSC experiments were performed with the liposomes comprising DMPC lipid, for which the main transition temperature should be observed at 24 °C (Silvius, 1982). The thermodynamic parameters of the thermograms are provided in Table 1. The

9

pre-transition point was not visible, and the main transition occurred at 22.1 °C for liposomes. The same lipid:DDMGlu molar ratio of 50:1 was investigated in all experiments. Similarly to fluorescence anisotropy changes, the thermodynamic parameters of the DMPC liposomes changed according to the DDM generation. DDM1Glu induced destabilization of the lipid bilayer. This process was indicated by the decrease in main transition temperature and enthalpy value. Both values were statistically significant in comparison to the control. Under the same conditions, DDM2Glu induced an increase in transition temperature (by 1.2 °C) as well as enthalpy value in comparison to the control. The temperature change was statistically significant, whereas the change in enthalpy value was not. DDM3Glu completely abolished phase transition.

3.3

Fluorescence quenching of HSA

The influence of DDM1-3Glu on HSA protein was studied by monitoring changes in protein fluorescence. The quenching of HSA fluorescence by DDMs is presented in Fig. 4. The overall effect of DDM1-3Glu on HSA fluorescence was not highly significant, with a maximum decrease in initial fluorescence by 35% observed for DDM2Glu. A roughly similar decrease (by 29%) was also observed in the case of DDM3Glu. The lowest fluorescence quenching of all DDM generations was observed in the case of DDM1Glu. In all cases, no changes in the position of the fluorescence emission maximum as a result of interaction were observed. Based on the results of HSA fluorescence quenching, the Stern-Volmer graphs were drawn, and a non-linear tendency was observed (Fig. 5). Downward Stern-Volmer curve is usually observed as a result of the fluorophore (here tryptophan) accessibility change in the course of increasing concentration of quencher (here DDM) (Lakowicz, 2006). From these, Stern-Volmer plots Ka and fa values (see Tab. 2) were calculated by using a modified Stern-Volmer equation (Lakowicz, 2006). The SternVolmer constants suggested that the highest interaction with HSA was shown by DDM2Glu, followed by DDM3Glu and DDM1Glu, respectively. Detailed analysis revealed that changes in Ka and fa of second- and third-generation DDMs as compared to first-generation DDMs were statistically significant. Comparison between second- and third-DDM generations showed a lack of significant change of fa.

3.4

Circular dichroism spectroscopy

The method allowed to monitor changes in the secondary structure of HSA protein with the increasing concentration of DDM1-3Glu. The circular dichroism spectra of HSA in the presence of DDMs are presented in Fig. 6. HSA secondary structures at different DDM1-3Glu concentrations 10

calculated by deconvolution are provided in Tab. 3. No changes in the secondary structure of HSA were observed for all investigated DDMs and concentrations.

4.

Discussion

Recent developments in the area of drug carriers have opened new possibilities to create more efficient systems for drug delivery. Among other types, the hyperbranched polymers represent a particularly promising group of drug carriers. It has been shown previously that there is no universal model of their interaction with biological systems and that the manner of interaction is associated with the structure of the core as well as the peripheral functional groups. As an example, it has been observed that surface-charged DDMs, in particular with cationic surface groups such as PAMAM, phosphorus-containing DDMs or cationic carbosilane DDMs (CSDDMs) (Ionov et al., 2012, 2011; Lombardo et al., 2016; Wrobel et al., 2014, 2012, 2011) had a highly strong influence on model lipid bilayer structure and properties. Only relatively few hyperbranched glycopolymers have been investigated with respect to their interactions with biological systems. Sugar-coated PPI and PEI hyperbranched polymers have so far been the most studied glyconanoparticles. Generally, hydrogen bonding has been postulated as the most important interaction between them and various biological systems (proteins and lipid model membranes), even in the presence of partially charged groups on the nanoparticle surface (Ciolkowski et al., 2012a, 2012b; Klajnert et al., 2008; Klementieva et al., 2013, 2011, Wrobel et al., 2017, 2015a). The extent of interactions has been observed to always increase with the number of surface groups and DDM generation. Relatively few research data exist regarding the interactions of carbosilane DDMs with biological models. Previous analyses demonstrated that cationic CS-DDMs were able to change the properties of the lipid bilayer in both hydrophobic and hydrophilic parts and decrease membrane fluidity (Wrobel et al., 2012). Moreover, it was also observed that the same DDMs induced the precipitation of negatively charged liposomes and also of biological membranes (Wrobel et al., 2014). The aggregation process was related to the electrostatic interaction between cationic surface groups of DDMs and anionic charges present on lipids. Recently, we have synthesized new types of first–third-generation carbosilane glycodendrimers (DDM1-3Glu) and tested their in vitro and in vivo toxicity on three model mammalian cell lines and zebrafish embryos (Liegertová et al., 2018). DDMGlu DDMs showed a very low toxic effect on a model cell line, but they also induced relatively high developmental toxicity in dechorionated zebrafish embryos. To further study the possible negative effects of DDM1-3Glu on living organisms, we 11

decided to monitor their influence on two different biological models: 1) neutral DMPC lipid model membranes (liposomes), which represent a biological membrane model and 2) human serum albumin as a protein model. Regarding the DDM-lipid membrane interactions, two independent methods were used for monitoring the process and extent of interaction: measurement of fluorescence anisotropy changes using DPH and TMA-DPH probes and differential scanning calorimetry. The results obtained from both methods were in good correlation. Generally, the extent of the interaction was not related to increasing DDM generation. The lipid membrane decreased its fluidity in the presence of high DDM concentrations of all generations studied. The highest influence (largest interaction) was observed in the case of DDM1Glu. A high impact on lipid membranes was also observed with DDM3Glu because it decreased the membrane fluidity in all tested concentrations. The lowest influence (weakest interaction) was induced by DDM2Glu, with only negligible changes in membrane rigidity being observed. Despite the similar tendencies observed for all DDMs, DDM1Glu displayed slightly different behaviour. At a certain range of low concentration (up to ≈ 3 µM), DDM1Glu increased membrane fluidity, as indicated by a decrease of anisotropy value. This observation was also confirmed by the DSC method. The transition temperature of DMPC liposomes decreased by 1 °C in the presence of DDM1Glu; this change signalled a membrane destabilization and increased membrane fluidity. In contrast, DDM2Glu caused a positive shift of the phase transition temperature by 1 °C compared to the control, an observation that provided evidence of membrane stabilization and increasing rigidity. DDM3Glu completely abolished phase transition in lipid bilayers, even at low concentrations, providing evidence of the strong impact of this DDM on the DMPC lipid bilayer. Interestingly, results similar to DDM1Glu were observed in our previous work (Wrobel et al., 2018) where cationic phosphonium carbosilane DDMs ((DmPMe3 and other derived types) interacted with the DMPC lipid bilayer. In this case, the DmPMe3 caused membrane destabilization and fluidity increase at relatively low DDM concentrations (up to ≈ 25 µM) (Wrobel et al., 2018). On the other hand, all DDMGlu generations interacted much more strongly and altered the DMPC membrane fluidity to a higher degree compared to DmPMe3 at higher DDM concentrations. Generally, all generations of DDMGlu decreased membrane fluidity at high concentrations but in contrast to other types of DDMs investigated elsewhere (Wrobel et al., 2018), this tendency was not related to increasing generation and number of surface groups. DDM1Glu induced the highest rigidness of the membrane as compared to the second and third generations of the same DDM. It 12

has already been described elsewhere that the influence of a DDM on bilayer structure becomes more significant with increasing size/generation. Generally, higher generations of the same type of DDM causes increased disturbances in lipid bilayers due to their larger size and higher affinity for lipids (more functional groups on the DDM surface) (Ionov et al., 2012; Wrobel et al., 2018, 2012, 2011). Our data indicate that this general rule need not always be valid. If our results were in agreement with this rule, the highest interaction with the lipid bilayers should be observed for the third-generation DDM, whereas the first generation should interact only negligibly. However, our results demonstrated that DDM1Glu induced the highest number of fluidity changes in lipid bilayers, whereas the lowest were observed for DDM2Glu. This abnormal behaviour of DDMGlu DDMs could be related to their behaviour in water solutions that has been observed previously. Experiments presented by Liegertova et al. showed that the hydrodynamic diameter of DDM1Glu was approx. 1 nm in a buffer solution, whereas the second- and third-generation DDMGlu DDMs created some larger structures/assemblies (Liegertová et al., 2018). DDM2Glu aggregates achieved sizes of tens up to several hundreds of nm, whereas DDM3Glu had an average diameter of 24 nm, suggesting more organized packing than DDM2Glu. The small hydrodynamic diameter of DDM1Glu as compared to larger DDM2Glu and DDM3Glu assemblies could be the main reason for the observed higher impact on neutral DMPC membranes. The hydrophobic interactions and hydrogen bonding through the glucose groups could be proposed as the main driving forces of interaction, as already described for other types of glycodendrimers (Ciolkowski et al., 2013, 2012b; Klajnert et al., 2008). The larger structures of DDM2-3Glu, lower accessibility of the hydrophobic part of these assemblies towards hydrophobic interaction with lipid membranes as well as lower effective concentration (of assemblies) could lead to a decreased possibility of interactions with liposomes. This hypothesis is further supported by the fact that larger assemblies of DDM2Glu showed lower interaction with lipid membranes than much smaller assemblies of DDM3Glu, mainly with the hydrophobic part of the lipid membrane (see Fig. 2). Another important piece of information was obtained from the comparison of the extent of the DDM1-3Glu interactions with the outer (TMA-DPH probe) and inner (DPH probe) space of the lipid bilayer. The largest changes in membrane fluidity were observed in the hydrophobic part of the bilayer (DPH probe), whereas fewer changes on the outer hydrophilic part of the lipid bilayer (TMA-DPH probe) were noted. Such results suggest that DDMs were able to interact/incorporate into the hydrophobic interior of the lipid membrane and change its fluidity. Therefore, besides hydrogen bonding, hydrophobic interactions seem to be much more important in the interaction process, mainly in the case of DDM1,3Glu. 13

The second part of the presented results is related to the interactions of DDM1-3Glu with the model protein HSA. HSA was chosen as a model protein due to its large abundance and significance in the human body. The fluorescence intensity of only one tryptophan situated in HSA protein is highly sensitive to any changes in its surroundings, and the shift in the position of the fluorescence maximum is related to polarity changes in the immediate vicinity of the chromophore (Lakowicz, 2006). Moreover, HSA was widely used for monitoring the interaction with various proteins and nanoparticles, including the cationic DDMs such as PAMAM, PPI or phosphorus-containing DDMs (Bakshi and Sood, 2004; Gabellieri et al., 2006; Jokiel et al., 2006; Klajnert et al., 2003; Klajnert and Bryszewska, 2002; Singh, 1998). It has also been shown that positively charged DDMs have a high impact on the structure of proteins and that the strength of interactions increased with higher DDM generations. As shown previously, hyperbranched glycopolymers such as maltose-modified PPI and PEI interacted with HSA to a significant degree (Klajnert et al., 2008). Surprisingly, no substantial difference was observed between PPI glycodendrimers and cationic PPI DDMs in the extent of interactions which was generation dependent, where higher generations of DDMs (third and fourth ones) showed an increased influence on HSA structure. Although maltose-modified PPI glycodendrimers had only a negligible influence on the secondary structure of another model protein, lysozyme; these glycodendrimers were able to decrease the accessibility of lysozyme’s tryptophan residues to quenchers. This observation led to the conclusion that maltose-modified PPI DDMs bind to the protein surface mainly via hydrogen bonding. A similar tendency was observed for maltose-modified hyperbranched PEI polymers (PEI-Mal) (Wrobel et al., 2017). It was shown that PEI-Mal was able to interact strongly with HSA and even change the secondary structure of the protein by both hydrogen bonding and electrostatic interactions. The interactions were sufficiently strong to create aggregates between PEI-Mal and HSA. The analysis of results presented in this article has demonstrated that the extent of interaction between HSA and DDM1-3Glu contrasts with the results obtained for liposomes. In general, fluorescence quenching experiments revealed that DDM2Glu had the highest impact on protein, whereas the least was observed for DDM1Glu. Based on measured fluorescence quenching, Stern-Volmer plots were drawn; however, rather than observing a linear relationship, downward curves were noted for all DDMs. Such curves suggest that the accessibility of tryptophan changes with increasing DDM concentration. This was further confirmed by calculation of Stern-Volmer constants from the modified Stern-Volmer equation. Fluorescence emission maxima were not changed in the presence of the DDMs; thus, the properties (polarity) of the close environment surrounding the tryptophan was not influenced. Interactions between 14

DDMGlu and HSA and their impact on protein secondary structure were also investigated using circular dichroism spectrometry. The results showed that DDMGlu DDMs did not change the secondary structure of HSA protein under any of the experimental conditions. Hence, protein fluorescence quenching is not related to protein molecule structural changes; DDMGlu could be attached to the protein without harmful effects on its structure and by this interaction, the fluorescence signal from Trp could be quenched. Similar conclusions were also drawn for maltose-modified PPI and PEI hyperbranched polymers (Ciolkowski et al., 2012a; Klajnert et al., 2008; Wrobel et al., 2017). On the other hand, the fluorescence quenching of HSA by DDMGlu was not related to DDM generation, as described for other types of hyperbranched glycopolymers (Klajnert et al., 2008; Wrobel et al., 2017). This may be explained in view of the results obtained for lipid bilayer interactions. It was shown previously that hyperbranched glycopolymers interact with proteins mainly via hydrogen bonding (Klajnert et al., 2008). We may expect similar interaction principles in the case of DDMGlu. It is therefore not surprising that a mirror-like extent of interaction was observed for both biological models. DDM1Glu interacted the most from all generations with the hydrophobic part of the lipid bilayer whereas with HSA, it showed the least interaction. Contrastingly, DDM2Glu demonstrated the fewest interactions from all generations with the hydrophobic part of the lipid bilayer; however, most likely due to the large assemblies in solution and many glucose units being available, it could create the most interactions with HSA via frequent hydrogen bonding. DDM3Glu interacted with a medium extent with both biological models. Based on our results, we suggest that the unusual generation-independent interaction extent of DDMGlu DDMs was related to the flexibility of the DDM molecule and its ability to expose its hydrophobic interior. -D-glucopyranosyl units present on DDMGlu are large surface groups and strongly influence the polymer properties. The DDM1Glu did not form any stable assemblies in the solution, and it possesses the lowest number of β-D-glucopyranosyl units. Therefore, we may expect the maximal flexibility of its backbone and the highest availability of the interior for hydrophobic interactions as compared to higher DDM generations. As a result, it easily interacted with the hydrophobic part of the lipid bilayer but only weakly with HSA. In contrast, the DDM2Glu created large assemblies in the solution, most likely stabilized by both the hydrophobic interactions and hydrogen bonding between the individual DDMs. The size of the resulting assembly, as well as the low flexibility, did not favour hydrophobic interactions with lipid bilayers, but on the other hand enabled strong interactions with HSA. Finally, the DDM 3Glu possesses the highest number of surface groups and is probably the most rigid structure. However, in comparison to DDM2Glu, it created only relatively small size assemblies in the buffer solution. 15

This may explain its moderate interactions with both the lipid bilayers and HSA, as compared to the other two DDMGlu generations. Hyperbranched polymers with glycoside modifications on their surface are promising nanoparticles in the development of drug delivery systems for anticancer therapy. It had already been shown that their toxicity in vitro and in vivo was significantly decreased, and despite their neutral surface groups, they were still able to interact with biological systems. Similar conclusions can be drawn for the glucose-modified carbosilane DDMs presented in this work. DDMGlu was able to interact with lipid bilayers and HSA by both hydrophobic and hydrogen bonding interactions. Interestingly, the extent of interaction was not dependent on DDM generation, and this property was related to the formation of DDM assemblies in solution. Relatively weak interaction with HSA protein without any sign of harmful influence on its secondary structure favours the potential use of such nanoparticles in in vivo applications. The formation of mid-size DDM assemblies, mainly of third-generation DDMGlu, could be used for the encapsulation of antitumor drugs and due to the presence of glucose units on the surface of such assemblies, these DDMs could be used to transport drugs into the interior of cancer cells overexpressing glucose transporters. Those properties make the DDMGlu a good candidate for the development of drug delivery systems for anticancer therapy.

5. Conclusions The interactions of first–third generations of glucose-modified carbosilane DDMs with neutral DMPC lipid model membranes and HSA were studied. The extent of interaction with both biological models was not related to the DDM generation. The highest level of interaction with HSA was demonstrated by DDM2Glu, with less interaction shown by DDM3Glu and the least by DDM1Glu. The opposite tendency was observed for the lipid bilayer model, where the highest amount of interaction was observed for DDM1Glu, followed by DDM3Glu and then DDM2Glu. Generally, all DDMGlus induced a decrease in membrane fluidity (increased rigidity) at higher concentrations and interacted only weakly with HSA. Based on our results, we suggested that the extent of interactions between DDMGlu DDMs and both biological models were related to the flexibility of the DDMGlu backbone, the availability of their hydrophobic interior for interaction as well as the formation of larger assemblies in a buffer solution. Due to the high level of interaction with lipid membranes, negligible influence on the model serum protein, theoretical possibility to actively target cancer cells via overexpressed glucose transporters, as well as due to other favourable properties described by us earlier (Liegertová et al., 2018) we may conclude that 16

the glucose-modified carbosilane DDMs represent good candidates for the development of safe and efficient drug delivery carriers in anti-cancer therapy.

Acknowledgements The authors acknowledge the assistance provided by the Research Infrastructure NanoEnviCz (Project

No.

LM2015073)

and

the

project

Pro-NanoEnviCz

(Reg.

No.

CZ.02.1.01/0.0/0.0/16_013/0001821), supported by the Ministry of Education, Youth and Sports of the Czech Republic and the European Union – European Structural and Investments Funds in the frame of the Operational Programme Research Development and Education, the ERDF/ESF project “UniQSurf - Centre of biointerfaces and hybrid functional materials" (No. CZ.02.1.01/0.0/0.0/17_048/0007411), the project No. UJEP-IGA-TC-2019-53-01-2 and the project COST LTC19049 supported by the Ministry of Education, Youth and Sports of the Czech Republic. This publication is based upon work from COST Action CA 17140 "Cancer Nanomedicine from the Bench to the Bedside" supported by COST (European Cooperation in Science and Technology).

17

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Figures

Figure 1.

Molecular structure of glucose-modified carbosilane dendrimers: A - first

generation (DDM1Glu), B - second generation (DDM2Glu), C - third generation (DDM3Glu).

Figure 2.

Anisotropy changes with increasing concentration of DDM1-3Glu. r - anisotropy

of fluorescence probe inside DMPC liposomes after DDM treatment, r0 - anisotropy of probe inside DMPC liposomes without DDM; A - DMPC model membranes with DPH probe; B DMPC model membranes with TMA-DPH probe. All data are expressed as mean ± S.E.M. n = 3; * p < 0.05 for each point vs control. Figure 3.

Thermograms of DMPC liposomes after DDMGlu addition.

Figure 4.

HSA quenching by DDMGlu dendrimers. (A) after DDM1Glu treatment; (B)

after DDM2Glu treatment; (C) after DDM3Glu treatment, ex = 290 nm, em = 310-500 nm.

Figure 5.

Stern–Volmer plots for HSA. Plots were constructed based on HSA fluorescence

quenching by DDMGlu DDMs, F0 - fluorescence intensity of pure HSA, Fn - fluorescence of HSA with increasing DDM concentration, ex = 290 nm, maximum of fluorescence intensity em = 349 nm. All data are expressed as mean ± S.D. n = 3; * p < 0.05 for each point vs control.

Figure 6.

CD spectra of HSA treated with DDM1-3Glu. (A) in the presence of DDM1Glu;

(B) in the presence of DDM2Glu; (C) in the presence of DDM3Glu.

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Figure(s)

Figure(s)

Figure(s)

Figure(s)

Figure(s)

Figure(s)

Tables Table 1.

DSC parameters of the DMPC liposome interaction with glucose modified

carbosilane dendrimers. All data were expressed as mean ± S.D., n = 3. Unpaired t-student test was used to compare samples values with control values. * p < 0.05 DDM generation

Tonset [°C]

H[J/g]

control

22.1±0.2

0.130±0.004

DDM1Glu

21.1±0.2*

0.061±0.010*

DDM2Glu

23.3±0.1*

0.137±0.006

DDM3Glu

-

-

Calorimetric parameters:

Tonset

- temperature at

which the thermal

effect

starts;

H – enthalpy.

Table 2.

Stern-Volmer constant (Ka) and the fraction of the initial fluorescence that is

accessible to quencher (fa). Both values were calculated from modified Stern–Volmer equation. All data were expressed as mean ± S.D., n = 3. Unpaired t-student test was used to compare samples: * - DDM2Glu and DDM3Glu values with DDM1Glu values; # DDM2Glu and DDM3Glu. * # p < 0.05

DDM generation

Ka x 104 [M-1]

fa

DDM1Glu

3.4±0.7

0.11±0.01

DDM2Glu

5.7±0.1*

0.25±0.01*

DDM3Glu

4.8±0.6* #

0.24±0.04*

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Table 3. 3Glu.

Composition of the secondary structures of HSA in the presence of DDM1-

Table shows percentage [%] of each secondary structure of proteins.

Dendrimer:protein molar ratio

control

1:2

1:1

2:1

6:1

10:1

15:1

20:1

-helix

69.1

68.8

68.8

68.8

68.7

68.8

68.8

69.0

-strand

9.2

9.2

9.2

9.2

9.2

9.2

9.2

9.2

random coil

21.7

22.0

22.0

22.0

22.1

22.1

22.0

21.8

-helix

69.1

69.1

68.7

69.0

69.0

69.0

69.0

69.0

-strand

9.2

9.2

9.2

9.2

9.2

9.2

9.2

9.2

random coil

21.7

21.7

22.1

21.8

21.8

21.8

21.8

21.8

-helix

69.1

69.1

69.1

69.0

69.0

69.9

68.8

68.8

-strand

9.2

9.2

9.2

9.2

9.2

9.2

9.2

9.2

random coil

21.7

21.7

21.7

21.8

21.8

21.9

22.0

22.0

first generation

second generation

third generation

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- degree of functional glucose surface groups and size of macromolecules affects interaction with proteins and model membranes - interaction with both biological models were not related with dendrimer generation - interaction with HSA showed DDM2Glu, lower DDM3Glu and the least DDM1Glu - the largest interaction with lipid bilayer was observed for DDM1Glu, lower for DDM3Glu and the least for DDM2Glu

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Authors contribution Dominika Wrobel: Conceptualization, Methodology, Formal analysis, Investigation, Writing Original Draft, Visualization. Monika Müllerová: Resources. Tomáš Strašák: Resources, Visualization. Květoslav Růžička: Formal analysis, Investigation. Michal Fulem: Investigation. Radka Kubíková: Investigation. Maria Bryszewska: Supervision, Writing - Review & Editing. Barbara Klajnert-Maculewicz: Supervision. Jan Malý Supervision, Project administration, Writing - Review & Editing.

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