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Interaction of phosphorus dendrimers with HIV peptides—Fluorescence studies of nano-complexes formation
Journal of Luminescence 148 (2014) 364–369
Contents lists available at ScienceDirect
Journal of Luminescence journal homepage: www.elsevier.com/locate/jlumin
Interaction of phosphorus dendrimers with HIV peptides—Fluorescence studies of nano-complexes formation Karol Ciepluch a,n, Maksim Ionov a, Jean-Pierre Majoral b, Maria Angeles Muñoz-Fernández c, Maria Bryszewska a a
Department of General Biophysics, Faculty of Biology and Environmental Protection, University of Lodz, Pomorska Street 141/143, 90-236 Lodz, Poland Laboratoire de Chimie de Coordination du CNRS (LCC), 205 Route de Narbonne, F-31077 Toulouse cedex 4, France c Laboratorio InmunoBiología Molecular, Hospital General Universitario Gregorio Marañón, Madrid, Spain b
art ic l e i nf o
a b s t r a c t
Article history: Received 17 October 2013 Received in revised form 11 December 2013 Accepted 20 December 2013 Available online 31 December 2013
In this study, dendrimers emerge as an alternative approach for delivery of HIV peptides to dendritic cells. Gp160, NH-EIDNYTNTIYTLLEE-COOH; P24, NH-DTINEEAAEW-COOH and Nef, NHGMDDPEREVLEWRFDSRLAFCOOH peptides were complexed with two types of positively charged phosphorus-containing dendrimers (CPD). Fluorescence polarization, dynamic light scattering, transmission and electron microscopy (TEM) techniques were chosen to evaluate the dendriplexes stability. We were able to show that complexes were stable in time and temperature. This is crucial for using these peptide/dendrimer nano-complexes in a new vaccine against HIV-1 infection. & 2013 Elsevier B.V. All rights reserved.
Keywords: Fluorescence polarization Nano-complex formation HIV peptides Phosphorus dendrimers Biophysical characteristics
1. Introduction Human immunodeficiency virus-1 (HIV-1) is a devastating human pathogen responsible for a worldwide pandemic of acquired immunodeficiency syndrome (AIDS). HIV infects vital cells in the human immune system such as helper T cells (specifically CD4þ T cells), macrophages, and dendritic cells. There is still a lack of an effective prophylactic or therapeutic vaccine despite extensive research on HIV and vaccine against this lentivirus. Vaccine efficacy depends on efficient delivery to professional antigen (Ag) presenting cells, such as dendritic cells. Therapy with dendritic cells can help to overcome diseases such as cancer or AIDS [1]. Antigenicity and adjuvanticity of vaccine components can be enhanced by encapsulation within nanoparticle (NP) vaccine carriers that are targeted to the human DC [2]. DCs regulate very important functions during HIV infection, for instance antibodies mediating neutralization, cytotoxicity and other antiviral reactivity. However, they are postulated to be the first cells to contact virus and they play a major role in establishing infection [3,4]. Captured virus can be transferred to CD4þ T cell, which are the primary target cell to infect [5]. To overcome this, it is desirable to transfer some antigen (e.g., HIV-derived peptides) to stimulate DCs. DCs can become a good immunotherapy vector but the choice of peptides is limited. Different types of systems as carriers for drugs
or biomacromolecules have been used including liposomes, nanoparticles, polymeric micelles, nanogels or dendrimers [6,7]. Dendrimers are considered as a new tool used in immunotherapy [8]. In this context, dendrimers emerge as an alternative approach for HIV peptide delivery to DCs. The main goal of this study was to demonstrate whether cationic phosphorus-containing dendrimers (Fig. 1) form complexes with HIV-derived peptides in various physico-chemical conditions and check whether these complexes are stable in time and in different temperatures. Cationic dendrimers interact efficiently with biomolecules, forming complexes by binding to the surface groups and they can also bind to HIV-derived peptides and the complex can be taken up by dendritic cells [9]. It is known that polycationic phosphorus dendrimers are able to penetrate the lipid membrane [10]. They possess a hydrophilic surface and a hydrophobic backbone and are well soluble in water, therefore they are potentially useful for various biological applications [11]. The results of this study can be helpful in creating a new strategy of immunotherapy of HIV-1 infection using the DCs loaded with synthetic HIV-derived peptides [12].
2. Material and methods 2.1. HIV-derived peptides and reagents
n
Corresponding author. E-mail address: [email protected] (K. Ciepluch).
0022-2313/$ - see front matter & 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jlumin.2013.12.049
Three different HIV-derived peptides unlabeled or labeled with fluorescein were synthesized in Eurogentec Company (Belgium).
K. Ciepluch et al. / Journal of Luminescence 148 (2014) 364–369
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Peptide derived from envelope Gp160 sequence, HIVHXB2 location Gp160 (634e648): NH-EIDNYTNTIYTLLEE-COOH, length 15 aminoacids charged ( 4); peptide derived from Gag-P24 sequence, HIVHXB2 location P24 (71e80): NH-DTINEEAAEW-COOH, length 10 amino-acids, charged ( 4) and peptide derived from Nef sequence, HIV-HXB2 location Nef (172–191): NHGMDDPEREVLEWRFDSRLAFCOOH, length 20 amino-acids, charged ( 3). All other reagents used were of analytical grade and purchased from Sigma-Aldrich Chemical Company. 2.2. Dendrimers Phosphorus dendrimers (CPD) were synthesized in the Laboratoire de Chimie de Coordination du CNRS, Toulouse, France. The main characteristics and synthesis of CPDs were described earlier [13]. CPDs-G3, C624H1104N183Cl48O42P45S42 (generation 3, 48 surface cationic end groups, MW: 16,280 g/mol; diameter: 4.1 nm) and CPDs-G4, C1296H2256N375Cl96O90P93S90 (generation 4, 96 surface cationic end groups, MW: 33 702g/mol; diameter: 5 nm), are presented in Fig. 1A and B. 2.3. Fluorescence polarization Samples were prepared by incubation of 0.5 μM peptide labeled with fluorescein with various concentrations of dendrimers in 10 mmol/L Na-phosphate buffer, pH 7.4 at a room temperature. Fluorescence polarization was measured using a Perkin Elmer LS-50B spectrofluorimeter (UK). Polarization was monitored at 520 nm (with a slit of 3 nm) with excitation at 495 nm (with a slit of 5 nm). Polarization is expressed as P ¼ ðI v GI H Þ=ðI v þ GI H Þ where IV and IH are the vertically and horizontally polarized emission intensities, respectively, when vertically polarized light is used to excite the sample, G is a g-factor (calculated automatically
Fig. 2. Changes in fluorescence polarization of the HIV-derived peptides P24, Gp160 and Nef on addition of cationic phosphorus dendrimers at increasing dendrimer/peptide molar ratios. Circles—CPD G3, squares—CPD G4. Peptide¼ 0.5 μmol/L, λex ¼ 497 nm, λem ¼ 532 nm, 0.01 mol/L, Na-phosphate buffer, pH 7.4. Results represent mean7 SD, n¼ 3.
Fig. 1. Structure of polycationic phosphorus dendrimers of generation 3 (A) and comparison between generation 3 (B) and generation 4 (C).
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by a Perkin Elmer software). The polarization of each sample was calculated from an average of 6 measurements each of IV and IH, and four values of such a set of averages were collected. 2.4. Transmission electron microscopy Complexes were formed by addition of dendrimers into peptides solutions at a dendrimer/peptide molar ratio of 3:1. The mixture was vortexed and incubated for 10 min at room temperature. Ten microliters of dendrimer/peptide mixture were placed on a copper grid with carbon surface for 10 min and dried with filter paper. The sample was stained with 2% (m/v) uranyl acetate for 2 min and dried. Transmission electron microscopy images were performed using JEOL-10 (Japan) transmission electron microscope. 2.5. Measurement of zeta potential and particle size The particle charge measurements were conducted using a phase analysis light scattering with Zetasizer Nano-ZS. The electrophoretic mobility of the dynamic light scattering samples of an applied electric field was measured in Malvern capillary plastic cells (DTS1061). Samples were prepared and measured at 25 1C in 10 mmol/L Na-phosphate buffer, pH 7.4 (buffer was filtered twice through 22 nm filter paper prior to use). Nine zeta potential measurements were collected for each dispersion, and the results were averaged. The zeta potential value was calculated directly from the Helmholtz–Smoluchowski equation using the Malvern software [14].
Fig. 3. The changes of zeta potential of HIV-derived peptides P24, Gp160, Nef upon addition of cationic phosphorus dendrimers. 10 mM phosphate buffer, pH 7.4, 24 1C. n¼ 3.
The particles size and size distribution (z-average mean) were measured using dynamic light scattering (DLS) in a photon correlation spectrometer (Zetasizer Nano-ZS, Malvern Instruments, UK). The refraction factor was assumed 1.33 while the detection angle was 901 and the wavelength was 633 nm. Samples in a 10 mmol/L Na-phosphate buffer, pH 7.4 were placed in the plastic cells DTS0012 (Malvern) and measured at 25 1C. The data were analyzed using the Malvern software. 2.6. Time- and temperature-stability of peptide–dendrimer complexes The stability and time of degradation of dendriplexes were measured using fluorescence polarization of fluorescein-labeled peptides incubated with dendrimers at a dendrimer/peptide molar ratio 3:1 for complexes of phosphorus dendrimers with peptides. Dendriplexes were prepared by incubation of 0.5 mM peptide labeled with fluorescein and dendrimers added in a suitable concentration. Experiments were carried out in room temperature (approx. 22 1C) and the change of fluorescence polarization in time was measured using a Perkin Elmer LS-50B spectrofluorimeter (UK). Polarization was monitored at 520 nm using peptides: P24 and Nef (with a bandwidth of 5 nm) with excitation at 495 nm (with a bandwidth of 7.5 nm). For Gp160 peptide conditions
Fig. 4. Average size of HIV-derived peptides P24—(A), Gp160—(B), Nef—(C) upon addition of cationic phosphorus dendrimers. 10 mM phosphate buffer, pH 7.4, 24 1C. n¼3.
K. Ciepluch et al. / Journal of Luminescence 148 (2014) 364–369
were: excitation—495 nm, emission—520 nm (bandwidths: 5, 7.5 respectively). Fluorescence polarization was measured in a temperature range from 27 1C to 80 1C. The time-dependent destruction of complexes leads to the decrease of fluorescence polarization degree of labeled peptides [15,16]. The polarization of each sample was calculated from an average of 6 measurements each of IV and IH, and three values of such a set of averages were collected.
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comparison with the respective controls (Fig. 3). The zeta potential curves reached a plateau at dendrimer/peptide molar ratios of (2):1. Particle size analysis by DLS indicated the formation of complexes between the CPD G3 and CPD G4 dendrimers and peptides. Upon addition of the dendrimers, the particle size increased up to 150–450 nm and the size of thus formed dendriplexes was stable above the dendrimer/peptide molar ratio of (2–3):1 (Fig. 4). These data lead to the conclusion that P24, Gp160 and Nef peptides can form complexes with both dendrimers in dendrimer/peptide molar ratios of (2–3):1.
3. Results 3.3. Transmission electron microscopy 3.1. Fluorescence polarization Fluorescence polarization of the fluorescein-labeled P24, Gp160 and Nef peptides in the presence and absence of cationic phosphorus dendrimers of generations 3 and 4 is shown in Fig. 2. The data show that gradually increasing concentrations of dendrimers increase the fluorescence polarization values of fluorescein labeled HIV peptides. Both generations of dendrimers (G3 and G4) form complexes with all kinds of studied HIV-peptides in 1:2–3 M ratio (peptide:dendrimer). 3.2. Average size and surface charge of the dendriplexes To correlate the dendrimer–peptide interaction observed by changes in fluorescence polarization with the variations in dendriplexes size and surface charge, DLS and zeta potential experiments were carried out on analogous samples that were used in the fluorescence experiments. Samples with the various dendrimer/peptide molar ratios (0.1–4):1 were prepared and Zeta average size and Zeta potential of dendriplexes were measured. The addition of dendrimers to the peptides in a range of (0.1–4): 1 (molar ratio) led to a significant increase in zeta potential in
The effect of dendrimers on the morphology of P24, Gp160 and Nef was followed by TEM. As it is demonstrated in electron micrographs (Fig. 6) during 10 min incubation with CPD G3 and CPD G4 the peptides formed numerous complexes. The images demonstrate the structure of complexed (Fig. 5. middle and right panels) and non-complexed (Fig. 6; left panels) peptides and indicate that presence of dendrimers in peptide suspension leads to the formation of dendrimer/peptide complex for all types of studied peptides. Complexes were formed when the dendrimer/ peptide molar ratio was 3:1. The size and shape distribution of the dendriplexes depended on the kind of the peptide. TEM images confirm that dendriplexes were formed that correlates with data obtained by other techniques described above. 3.4. Dendriplexes stability in time The time dependent fluorescence polarization values of fluorescein-labeled peptides complexed with dendrimers are shown in Fig. 6. The complexes of phosphorus dendrimers with all peptides decayed after 6 h of incubation. However, the complete decay of the complex was observed after 48 h only.
Fig. 5. Electron micrographs of dendrimer/peptide mixtures composed of CPD G3 or CPD G4 dendrimers and HIV-derived peptides Gp160, P24 and Nef. The molar ratio of a dendrimer/peptide was 3:1. A magnification of 30.000–50.000 was used to examine the peptides and their complexes with dendrimers. Bar ¼ 200 nm.
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Fig. 6. Changes of fluorescence polarization of fluorescein labeled HIV-derived peptides, P24, Gp160, Nef in the presence of phosphorus dendrimers at dendrimer/ peptide molar ratio 3:1.
3.5. Temperature-stability of dendriplexes To illustrate the temperature-dependent stability of dendriplexes, the fluorescent polarization technique was used. The data presented in Fig. 7 reflect the changes of fluorescence polarization values of fluorescein labeled P24, Gp160 and Nef peptides at increasing temperatures from 27 to 60–80 1C. Dendriplexes formed with phosphorus dendrimers were quite stable in temperatures below 50 1C. Above this temperature, complexes of phosphorus dendrimers with Nef and Gp160 peptides decayed significantly while the complexes with the smallest peptide (P24) were stable in the whole range of temperatures.
4. Discussion It is known that HIV-1 mainly infects CD4 þ T cells but can also replicate in other cells such as macrophages and dendritic cells (DCs) [17,18]. The contact between DCs and CD4 þ T cells has been shown to promote a very efficient transmission of HIV-1. Our idea was that dendrimers permit a better uptake of HIV antigens to DCs, which are efficient in presenting these antigens to T cells, therefore dendrimers can be a great tool in immunotherapy. The choice of available dendrimers is essential. As dendrimers were successfully used as transfecting agents [19–21], we decided to test some types of dendrimers for their ability to carry HIV peptides. Fluorescence spectroscopy measurements were firstly conducted in order to estimate the interaction of phosphorus dendrimers with
Fig. 7. Changes of fluorescence polarization of fluorescein labeled HIV-derived peptides, P24, Gp160, Nef in the presence of phosphorus dendrimers in increasing temperatures. Dendrimer/peptide molar ratio was 3:1.
P24, Gp160 and Nef peptides. The increase in the fluorescence polarization values is an indication of a decrease in mobility of the probe molecules attached to one of the peptide termini as a result of the binding of dendrimer molecules to the peptide. These findings suggest that dendrimers interact with peptide molecules and form complexes in studied concentration range. Formation of dendriplexes means that electrostatic interactions take place between positively charged dendrimers and anionic HIV peptides. To correlate the dendrimer–peptide interaction observed by changes in fluorescence polarization with the variations in dendriplexes size and surface charge, DLS and zeta potential experiments were carried out on analogous samples that were used in the fluorescence experiments. The zeta potential curves reached a plateau at dendrimer/ peptide molar ratios of (2):1. Particle size analysis by DLS indicated also the formation of complexes between the dendrimers and peptides. Upon addition of the dendrimer (G3), the particle size increased up to 130– 300 nm and the size of thus formed dendriplexes did not change for a dendrimer/peptide molar ratio of (2–3):1. Further increase of the dendrimer concentration did not change the dendriplexes size. These data lead to the conclusion that P24, Gp160 and Nef peptides can form complexes with both dendrimers in a dendrimer/peptide molar ratios (2–3):1. For 2–3/1 CPD/peptide molar ratio the complex is fully saturated by dendrimers. For the
K. Ciepluch et al. / Journal of Luminescence 148 (2014) 364–369
dendrimer of generation G4 we observed much bigger size from 250 to 400 nm. The effect of dendrimers on the morphology of P24, Gp160 and Nef was followed by TEM. As it is demonstrated in electron micrographs, during 10 min incubation with dendrimers the peptides formed numerous complexes corresponding to the previously reported nano-complexes with carbosilane dendrimers [22]. The images show the structure of complexed and noncomplexed peptides and indicate that the presence of dendrimers in peptide suspension leads to the formation of dendrimer/peptide complexes for all types of studied peptides:130–200 nm complexes were formed when the dendrimer/peptide molar ratio was 3:1. Anyway, the size and shape distribution of the dendriplexes depended on the kind of the considered peptide. Since the preparation of the samples for TEM requires the solvent evaporation from the peptide/dendrimer mixture, such used treatment may not reflect the exact particle distribution present in solution. Despite this, TEM images confirm that dendriplexes were formed corroborating the data obtained by the other techniques described above. We have demonstrated that the peptide/dendrimer complexes stability depends on time, temperature. In a previous work [16] we have shown the time stability of dendriplexes formed by carbosilane dendrimers and P24, Gp160 and Nef peptides. Indeed dendriplexes formed by BDBR0011 (cationic carbosilane dendrimer with Si–C bonds) were more stable than those containing NN16 (cationic carbosilane dendrimer with less stable Si–O bonds), 50 and 2 h, respectively. However, the long life time of such complexes might not be good due to toxicity of dendrimers. So, we decided to use phosphorus dendrimers. The complexes formed by phosphorus dendrimers with peptides decayed after 6 h. It is also important that such complexes are stable in temperatures close to physiological (37–40 1C). All tested complexes decayed at higher temperatures between 50 1C and 60 1C, except for P24þ CPD. P24 is the smallest peptide (10 amino acids) and it is likely that the conformation of this peptide allows to create more stable complex with dendrimers. It is also known that dendrimers can bind to genetic material (e.g., siRNA) and protect it against proteins in bloodstream which is extremely important in the context of therapy using nanocarriers or in creating vaccine [23]. If a size of a dendriplex increases it may mean that aggregation takes place. The sizes of complexes of phosphorus dendrimers with peptides are near 200–300 nm. The size of approximately 200 nm is optimal for crossing the membrane barrier. The stability conditions for all vectors delivering their load to a cell are crucial. Therefore, the time- and temperature-stability must be checked before transfection experiments. These observations allow us to think that dendrimers could be used as a tool in anti-HIV immunotherapy. 5. Conclusion Obtained results show that the water-soluble cationic dendrimers can be considered for delivery of HIV peptides to dendritic
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cells. Two generations of phosphorus dendrimers were selected for interacting with HIV peptides: stability of the resulting dendriplexes was checked in different conditions. All tested dendrimers effectively formed the complexes with HIV-derived peptides Gp160, P24 and Nef. They were time-, temperature, which is a feature needed for the effective uptake by dendritic cells (DC). However only dendrimer generation G3 creates complex with size is optimal for membrane crossing. These results convince us that using these dendrimers in studies on a vaccine against HIV infection is of strong interest, as they have all important features which are necessary for anti-HIV vaccine candidates.
Acknowledgments Studies were funded by the Project no. 1/EuroNanoMed/2010, DENPEPTHIV financed by National Research and Development Center, Poland (PS09102669, FIS (PI08222), CIBER-BBN).
References [1] D. Banerjee, P.E. Matthews, E. Matayeva, J.L. Kaufman, R.M. Steinman, K.M. Dhodapkar, J. Immunother. 31 (2008) 113. [2] P.J. Tacken, I.J. de Vries, R. Torensma, C.G. Figdor, Nat. Rev. Immunol. 7 (2007) 790. [3] G. Schuler, Eur. J. Immunol. 40 (2010) 2123. [4] R.M. Steinman, J. Banchereau, Nature 449 (2007) 419. [5] D. McDonald, L. Wu, S.M. Bohks, V.N. KewalRamani, D. Unutmaz, T.J. Hope, Science 300 (2003) 1295. [6] H.M. Aliabadi, A. Lavasanifar, Expert Opin. Drug Deliv. 3 (2006) 139. [7] K. Gardikis, D. Fessas, M. Signorelli, K. Dimas, C. Tsimplouli, M. Ionov, C. Demetzos, J. Nanosci. Nanotechnol. 11 (2011) 3764. [8] K. Hulikova, J. Svoboda, V. Benson, V. Grobarova, A. Fiserova, Int. Immunopharmacol. 8 (2011) 955. [9] M. Pion, M.J. Serramia, L. Diaz, M. Bryszewska, T. Gallart, F. Garcia, R. Gomez, F.J. de la Mata, M.A. Munoz-Fernandez, Biomaterials 31 (2010) 8749. [10] M. Ionov, D. Wróbel, K. Gardikis, S. Hatziantoniou, C. Demetzos, J.P. Majoral, B. Klajnert, M. Bryszewska, Chem. Phys. Lipids 165 (2012) 408. [11] J. Solassol, C. Crozet, V. Perrier, J. Leclaire, F. Béranger, A.M. Caminade, B. Meunier, D. Dormont, J.P. Majoral, S. Lehmann, J. Gen. Virol. 85 (2004) 1791. [12] J.F. Bermejo, P. Ortega, L. Chonco, R. Eritja, R. Samaniego, M. Mullner, E. DeJesus, F.J. De la Mata, J.C. Flores, R. Gomez, M.A. Munoz-Fernandez, Chem. Eur. J. 13 (2007) 483. [13] A.M. Caminade, J.P. Majoral, Prog. Polym. Sci. 30 (2005) 491. [14] M. Smoluchowski, in: L. Graetz (Ed.), Handbuch der Elektrizität und de Magnetismus, 2nd ed.,Barth, Verlag, Leipzig1921, p. 366. [15] D. Shcharbin, E. Pedziwiatr, M. Bryszewska, J. Controll. Release 135 (2009) 186. [16] M.S. Nasir, M.E. Jolley, High Throughput Screen. 2 (1999) 177. [17] C.M. Coleman, L. Wu, Retrovirology 6 (2009) 51. [18] L. Wu, V.N. KewalRamani, Nat. Rev. Immunol. 6 (2006) 859. [19] J. Haensler, F.C. Szoka, Bioconjugate Chem. 4 (1993) 372. [20] C. Loup, M.A. Zanta, A.M. Caminade, J.P. Majoral, B. Meunier, Chem.—Eur. J. 5 (1999) 3644. [21] A.V. Maksimenko, V. Mandrouguine, M.B. Gottikh, J.R. Bertrand, J.P. Majoral, C. Malvy, J. Gen. Med. 5 (2003) 61. [22] M. Ionov, K. Ciepluch, B. Klajnert, S. Glińska, R. Gomez-Ramirez, F.J. de la Mata, M.A. Munoz-Fernandez, M. Bryszewska, Colloids Surf. B: Biointerfaces 101 (2013) 236. [23] L. Chonco, J.F. Bermejo-Martín, P. Ortega, D. Shcharbin, E. Pedziwiatr, B. Klajnert, F.J. De la Mata, R. Eritja, R. Gómez, M. Bryszewska, M.A. MuñozFernandez, Org. Biomol. Chem. 5 (2007) 1886.
Contents lists available at ScienceDirect
Journal of Luminescence journal homepage: www.elsevier.com/locate/jlumin
Interaction of phosphorus dendrimers with HIV peptides—Fluorescence studies of nano-complexes formation Karol Ciepluch a,n, Maksim Ionov a, Jean-Pierre Majoral b, Maria Angeles Muñoz-Fernández c, Maria Bryszewska a a
Department of General Biophysics, Faculty of Biology and Environmental Protection, University of Lodz, Pomorska Street 141/143, 90-236 Lodz, Poland Laboratoire de Chimie de Coordination du CNRS (LCC), 205 Route de Narbonne, F-31077 Toulouse cedex 4, France c Laboratorio InmunoBiología Molecular, Hospital General Universitario Gregorio Marañón, Madrid, Spain b
art ic l e i nf o
a b s t r a c t
Article history: Received 17 October 2013 Received in revised form 11 December 2013 Accepted 20 December 2013 Available online 31 December 2013
In this study, dendrimers emerge as an alternative approach for delivery of HIV peptides to dendritic cells. Gp160, NH-EIDNYTNTIYTLLEE-COOH; P24, NH-DTINEEAAEW-COOH and Nef, NHGMDDPEREVLEWRFDSRLAFCOOH peptides were complexed with two types of positively charged phosphorus-containing dendrimers (CPD). Fluorescence polarization, dynamic light scattering, transmission and electron microscopy (TEM) techniques were chosen to evaluate the dendriplexes stability. We were able to show that complexes were stable in time and temperature. This is crucial for using these peptide/dendrimer nano-complexes in a new vaccine against HIV-1 infection. & 2013 Elsevier B.V. All rights reserved.
Keywords: Fluorescence polarization Nano-complex formation HIV peptides Phosphorus dendrimers Biophysical characteristics
1. Introduction Human immunodeficiency virus-1 (HIV-1) is a devastating human pathogen responsible for a worldwide pandemic of acquired immunodeficiency syndrome (AIDS). HIV infects vital cells in the human immune system such as helper T cells (specifically CD4þ T cells), macrophages, and dendritic cells. There is still a lack of an effective prophylactic or therapeutic vaccine despite extensive research on HIV and vaccine against this lentivirus. Vaccine efficacy depends on efficient delivery to professional antigen (Ag) presenting cells, such as dendritic cells. Therapy with dendritic cells can help to overcome diseases such as cancer or AIDS [1]. Antigenicity and adjuvanticity of vaccine components can be enhanced by encapsulation within nanoparticle (NP) vaccine carriers that are targeted to the human DC [2]. DCs regulate very important functions during HIV infection, for instance antibodies mediating neutralization, cytotoxicity and other antiviral reactivity. However, they are postulated to be the first cells to contact virus and they play a major role in establishing infection [3,4]. Captured virus can be transferred to CD4þ T cell, which are the primary target cell to infect [5]. To overcome this, it is desirable to transfer some antigen (e.g., HIV-derived peptides) to stimulate DCs. DCs can become a good immunotherapy vector but the choice of peptides is limited. Different types of systems as carriers for drugs
or biomacromolecules have been used including liposomes, nanoparticles, polymeric micelles, nanogels or dendrimers [6,7]. Dendrimers are considered as a new tool used in immunotherapy [8]. In this context, dendrimers emerge as an alternative approach for HIV peptide delivery to DCs. The main goal of this study was to demonstrate whether cationic phosphorus-containing dendrimers (Fig. 1) form complexes with HIV-derived peptides in various physico-chemical conditions and check whether these complexes are stable in time and in different temperatures. Cationic dendrimers interact efficiently with biomolecules, forming complexes by binding to the surface groups and they can also bind to HIV-derived peptides and the complex can be taken up by dendritic cells [9]. It is known that polycationic phosphorus dendrimers are able to penetrate the lipid membrane [10]. They possess a hydrophilic surface and a hydrophobic backbone and are well soluble in water, therefore they are potentially useful for various biological applications [11]. The results of this study can be helpful in creating a new strategy of immunotherapy of HIV-1 infection using the DCs loaded with synthetic HIV-derived peptides [12].
2. Material and methods 2.1. HIV-derived peptides and reagents
n
Corresponding author. E-mail address: [email protected] (K. Ciepluch).
0022-2313/$ - see front matter & 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jlumin.2013.12.049
Three different HIV-derived peptides unlabeled or labeled with fluorescein were synthesized in Eurogentec Company (Belgium).
K. Ciepluch et al. / Journal of Luminescence 148 (2014) 364–369
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Peptide derived from envelope Gp160 sequence, HIVHXB2 location Gp160 (634e648): NH-EIDNYTNTIYTLLEE-COOH, length 15 aminoacids charged ( 4); peptide derived from Gag-P24 sequence, HIVHXB2 location P24 (71e80): NH-DTINEEAAEW-COOH, length 10 amino-acids, charged ( 4) and peptide derived from Nef sequence, HIV-HXB2 location Nef (172–191): NHGMDDPEREVLEWRFDSRLAFCOOH, length 20 amino-acids, charged ( 3). All other reagents used were of analytical grade and purchased from Sigma-Aldrich Chemical Company. 2.2. Dendrimers Phosphorus dendrimers (CPD) were synthesized in the Laboratoire de Chimie de Coordination du CNRS, Toulouse, France. The main characteristics and synthesis of CPDs were described earlier [13]. CPDs-G3, C624H1104N183Cl48O42P45S42 (generation 3, 48 surface cationic end groups, MW: 16,280 g/mol; diameter: 4.1 nm) and CPDs-G4, C1296H2256N375Cl96O90P93S90 (generation 4, 96 surface cationic end groups, MW: 33 702g/mol; diameter: 5 nm), are presented in Fig. 1A and B. 2.3. Fluorescence polarization Samples were prepared by incubation of 0.5 μM peptide labeled with fluorescein with various concentrations of dendrimers in 10 mmol/L Na-phosphate buffer, pH 7.4 at a room temperature. Fluorescence polarization was measured using a Perkin Elmer LS-50B spectrofluorimeter (UK). Polarization was monitored at 520 nm (with a slit of 3 nm) with excitation at 495 nm (with a slit of 5 nm). Polarization is expressed as P ¼ ðI v GI H Þ=ðI v þ GI H Þ where IV and IH are the vertically and horizontally polarized emission intensities, respectively, when vertically polarized light is used to excite the sample, G is a g-factor (calculated automatically
Fig. 2. Changes in fluorescence polarization of the HIV-derived peptides P24, Gp160 and Nef on addition of cationic phosphorus dendrimers at increasing dendrimer/peptide molar ratios. Circles—CPD G3, squares—CPD G4. Peptide¼ 0.5 μmol/L, λex ¼ 497 nm, λem ¼ 532 nm, 0.01 mol/L, Na-phosphate buffer, pH 7.4. Results represent mean7 SD, n¼ 3.
Fig. 1. Structure of polycationic phosphorus dendrimers of generation 3 (A) and comparison between generation 3 (B) and generation 4 (C).
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by a Perkin Elmer software). The polarization of each sample was calculated from an average of 6 measurements each of IV and IH, and four values of such a set of averages were collected. 2.4. Transmission electron microscopy Complexes were formed by addition of dendrimers into peptides solutions at a dendrimer/peptide molar ratio of 3:1. The mixture was vortexed and incubated for 10 min at room temperature. Ten microliters of dendrimer/peptide mixture were placed on a copper grid with carbon surface for 10 min and dried with filter paper. The sample was stained with 2% (m/v) uranyl acetate for 2 min and dried. Transmission electron microscopy images were performed using JEOL-10 (Japan) transmission electron microscope. 2.5. Measurement of zeta potential and particle size The particle charge measurements were conducted using a phase analysis light scattering with Zetasizer Nano-ZS. The electrophoretic mobility of the dynamic light scattering samples of an applied electric field was measured in Malvern capillary plastic cells (DTS1061). Samples were prepared and measured at 25 1C in 10 mmol/L Na-phosphate buffer, pH 7.4 (buffer was filtered twice through 22 nm filter paper prior to use). Nine zeta potential measurements were collected for each dispersion, and the results were averaged. The zeta potential value was calculated directly from the Helmholtz–Smoluchowski equation using the Malvern software [14].
Fig. 3. The changes of zeta potential of HIV-derived peptides P24, Gp160, Nef upon addition of cationic phosphorus dendrimers. 10 mM phosphate buffer, pH 7.4, 24 1C. n¼ 3.
The particles size and size distribution (z-average mean) were measured using dynamic light scattering (DLS) in a photon correlation spectrometer (Zetasizer Nano-ZS, Malvern Instruments, UK). The refraction factor was assumed 1.33 while the detection angle was 901 and the wavelength was 633 nm. Samples in a 10 mmol/L Na-phosphate buffer, pH 7.4 were placed in the plastic cells DTS0012 (Malvern) and measured at 25 1C. The data were analyzed using the Malvern software. 2.6. Time- and temperature-stability of peptide–dendrimer complexes The stability and time of degradation of dendriplexes were measured using fluorescence polarization of fluorescein-labeled peptides incubated with dendrimers at a dendrimer/peptide molar ratio 3:1 for complexes of phosphorus dendrimers with peptides. Dendriplexes were prepared by incubation of 0.5 mM peptide labeled with fluorescein and dendrimers added in a suitable concentration. Experiments were carried out in room temperature (approx. 22 1C) and the change of fluorescence polarization in time was measured using a Perkin Elmer LS-50B spectrofluorimeter (UK). Polarization was monitored at 520 nm using peptides: P24 and Nef (with a bandwidth of 5 nm) with excitation at 495 nm (with a bandwidth of 7.5 nm). For Gp160 peptide conditions
Fig. 4. Average size of HIV-derived peptides P24—(A), Gp160—(B), Nef—(C) upon addition of cationic phosphorus dendrimers. 10 mM phosphate buffer, pH 7.4, 24 1C. n¼3.
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were: excitation—495 nm, emission—520 nm (bandwidths: 5, 7.5 respectively). Fluorescence polarization was measured in a temperature range from 27 1C to 80 1C. The time-dependent destruction of complexes leads to the decrease of fluorescence polarization degree of labeled peptides [15,16]. The polarization of each sample was calculated from an average of 6 measurements each of IV and IH, and three values of such a set of averages were collected.
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comparison with the respective controls (Fig. 3). The zeta potential curves reached a plateau at dendrimer/peptide molar ratios of (2):1. Particle size analysis by DLS indicated the formation of complexes between the CPD G3 and CPD G4 dendrimers and peptides. Upon addition of the dendrimers, the particle size increased up to 150–450 nm and the size of thus formed dendriplexes was stable above the dendrimer/peptide molar ratio of (2–3):1 (Fig. 4). These data lead to the conclusion that P24, Gp160 and Nef peptides can form complexes with both dendrimers in dendrimer/peptide molar ratios of (2–3):1.
3. Results 3.3. Transmission electron microscopy 3.1. Fluorescence polarization Fluorescence polarization of the fluorescein-labeled P24, Gp160 and Nef peptides in the presence and absence of cationic phosphorus dendrimers of generations 3 and 4 is shown in Fig. 2. The data show that gradually increasing concentrations of dendrimers increase the fluorescence polarization values of fluorescein labeled HIV peptides. Both generations of dendrimers (G3 and G4) form complexes with all kinds of studied HIV-peptides in 1:2–3 M ratio (peptide:dendrimer). 3.2. Average size and surface charge of the dendriplexes To correlate the dendrimer–peptide interaction observed by changes in fluorescence polarization with the variations in dendriplexes size and surface charge, DLS and zeta potential experiments were carried out on analogous samples that were used in the fluorescence experiments. Samples with the various dendrimer/peptide molar ratios (0.1–4):1 were prepared and Zeta average size and Zeta potential of dendriplexes were measured. The addition of dendrimers to the peptides in a range of (0.1–4): 1 (molar ratio) led to a significant increase in zeta potential in
The effect of dendrimers on the morphology of P24, Gp160 and Nef was followed by TEM. As it is demonstrated in electron micrographs (Fig. 6) during 10 min incubation with CPD G3 and CPD G4 the peptides formed numerous complexes. The images demonstrate the structure of complexed (Fig. 5. middle and right panels) and non-complexed (Fig. 6; left panels) peptides and indicate that presence of dendrimers in peptide suspension leads to the formation of dendrimer/peptide complex for all types of studied peptides. Complexes were formed when the dendrimer/ peptide molar ratio was 3:1. The size and shape distribution of the dendriplexes depended on the kind of the peptide. TEM images confirm that dendriplexes were formed that correlates with data obtained by other techniques described above. 3.4. Dendriplexes stability in time The time dependent fluorescence polarization values of fluorescein-labeled peptides complexed with dendrimers are shown in Fig. 6. The complexes of phosphorus dendrimers with all peptides decayed after 6 h of incubation. However, the complete decay of the complex was observed after 48 h only.
Fig. 5. Electron micrographs of dendrimer/peptide mixtures composed of CPD G3 or CPD G4 dendrimers and HIV-derived peptides Gp160, P24 and Nef. The molar ratio of a dendrimer/peptide was 3:1. A magnification of 30.000–50.000 was used to examine the peptides and their complexes with dendrimers. Bar ¼ 200 nm.
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Fig. 6. Changes of fluorescence polarization of fluorescein labeled HIV-derived peptides, P24, Gp160, Nef in the presence of phosphorus dendrimers at dendrimer/ peptide molar ratio 3:1.
3.5. Temperature-stability of dendriplexes To illustrate the temperature-dependent stability of dendriplexes, the fluorescent polarization technique was used. The data presented in Fig. 7 reflect the changes of fluorescence polarization values of fluorescein labeled P24, Gp160 and Nef peptides at increasing temperatures from 27 to 60–80 1C. Dendriplexes formed with phosphorus dendrimers were quite stable in temperatures below 50 1C. Above this temperature, complexes of phosphorus dendrimers with Nef and Gp160 peptides decayed significantly while the complexes with the smallest peptide (P24) were stable in the whole range of temperatures.
4. Discussion It is known that HIV-1 mainly infects CD4 þ T cells but can also replicate in other cells such as macrophages and dendritic cells (DCs) [17,18]. The contact between DCs and CD4 þ T cells has been shown to promote a very efficient transmission of HIV-1. Our idea was that dendrimers permit a better uptake of HIV antigens to DCs, which are efficient in presenting these antigens to T cells, therefore dendrimers can be a great tool in immunotherapy. The choice of available dendrimers is essential. As dendrimers were successfully used as transfecting agents [19–21], we decided to test some types of dendrimers for their ability to carry HIV peptides. Fluorescence spectroscopy measurements were firstly conducted in order to estimate the interaction of phosphorus dendrimers with
Fig. 7. Changes of fluorescence polarization of fluorescein labeled HIV-derived peptides, P24, Gp160, Nef in the presence of phosphorus dendrimers in increasing temperatures. Dendrimer/peptide molar ratio was 3:1.
P24, Gp160 and Nef peptides. The increase in the fluorescence polarization values is an indication of a decrease in mobility of the probe molecules attached to one of the peptide termini as a result of the binding of dendrimer molecules to the peptide. These findings suggest that dendrimers interact with peptide molecules and form complexes in studied concentration range. Formation of dendriplexes means that electrostatic interactions take place between positively charged dendrimers and anionic HIV peptides. To correlate the dendrimer–peptide interaction observed by changes in fluorescence polarization with the variations in dendriplexes size and surface charge, DLS and zeta potential experiments were carried out on analogous samples that were used in the fluorescence experiments. The zeta potential curves reached a plateau at dendrimer/ peptide molar ratios of (2):1. Particle size analysis by DLS indicated also the formation of complexes between the dendrimers and peptides. Upon addition of the dendrimer (G3), the particle size increased up to 130– 300 nm and the size of thus formed dendriplexes did not change for a dendrimer/peptide molar ratio of (2–3):1. Further increase of the dendrimer concentration did not change the dendriplexes size. These data lead to the conclusion that P24, Gp160 and Nef peptides can form complexes with both dendrimers in a dendrimer/peptide molar ratios (2–3):1. For 2–3/1 CPD/peptide molar ratio the complex is fully saturated by dendrimers. For the
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dendrimer of generation G4 we observed much bigger size from 250 to 400 nm. The effect of dendrimers on the morphology of P24, Gp160 and Nef was followed by TEM. As it is demonstrated in electron micrographs, during 10 min incubation with dendrimers the peptides formed numerous complexes corresponding to the previously reported nano-complexes with carbosilane dendrimers [22]. The images show the structure of complexed and noncomplexed peptides and indicate that the presence of dendrimers in peptide suspension leads to the formation of dendrimer/peptide complexes for all types of studied peptides:130–200 nm complexes were formed when the dendrimer/peptide molar ratio was 3:1. Anyway, the size and shape distribution of the dendriplexes depended on the kind of the considered peptide. Since the preparation of the samples for TEM requires the solvent evaporation from the peptide/dendrimer mixture, such used treatment may not reflect the exact particle distribution present in solution. Despite this, TEM images confirm that dendriplexes were formed corroborating the data obtained by the other techniques described above. We have demonstrated that the peptide/dendrimer complexes stability depends on time, temperature. In a previous work [16] we have shown the time stability of dendriplexes formed by carbosilane dendrimers and P24, Gp160 and Nef peptides. Indeed dendriplexes formed by BDBR0011 (cationic carbosilane dendrimer with Si–C bonds) were more stable than those containing NN16 (cationic carbosilane dendrimer with less stable Si–O bonds), 50 and 2 h, respectively. However, the long life time of such complexes might not be good due to toxicity of dendrimers. So, we decided to use phosphorus dendrimers. The complexes formed by phosphorus dendrimers with peptides decayed after 6 h. It is also important that such complexes are stable in temperatures close to physiological (37–40 1C). All tested complexes decayed at higher temperatures between 50 1C and 60 1C, except for P24þ CPD. P24 is the smallest peptide (10 amino acids) and it is likely that the conformation of this peptide allows to create more stable complex with dendrimers. It is also known that dendrimers can bind to genetic material (e.g., siRNA) and protect it against proteins in bloodstream which is extremely important in the context of therapy using nanocarriers or in creating vaccine [23]. If a size of a dendriplex increases it may mean that aggregation takes place. The sizes of complexes of phosphorus dendrimers with peptides are near 200–300 nm. The size of approximately 200 nm is optimal for crossing the membrane barrier. The stability conditions for all vectors delivering their load to a cell are crucial. Therefore, the time- and temperature-stability must be checked before transfection experiments. These observations allow us to think that dendrimers could be used as a tool in anti-HIV immunotherapy. 5. Conclusion Obtained results show that the water-soluble cationic dendrimers can be considered for delivery of HIV peptides to dendritic
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cells. Two generations of phosphorus dendrimers were selected for interacting with HIV peptides: stability of the resulting dendriplexes was checked in different conditions. All tested dendrimers effectively formed the complexes with HIV-derived peptides Gp160, P24 and Nef. They were time-, temperature, which is a feature needed for the effective uptake by dendritic cells (DC). However only dendrimer generation G3 creates complex with size is optimal for membrane crossing. These results convince us that using these dendrimers in studies on a vaccine against HIV infection is of strong interest, as they have all important features which are necessary for anti-HIV vaccine candidates.
Acknowledgments Studies were funded by the Project no. 1/EuroNanoMed/2010, DENPEPTHIV financed by National Research and Development Center, Poland (PS09102669, FIS (PI08222), CIBER-BBN).
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