Journal of Photochemistry and Photobiology B: Biology 130 (2014) 30–39
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Encapsulation of biogenic and synthetic polyamines by nanoparticles PEG and mPEG-anthracene S. Sanyakamdhorn, P. Chanphai, H.A. Tajmir-Riahi ⇑ Department of Chemistry–Physics, University of Québec in Trois-Rivières, C.P. 500, Trois-Rivières, Québec G9A 5H7, Canada
a r t i c l e
i n f o
Article history: Received 30 September 2013 Received in revised form 20 October 2013 Accepted 22 October 2013 Available online 1 November 2013 Keywords: PEG Polymer Polyamine Binding site FTIR Fluorescence spectroscopy
a b s t r a c t Synthetic polymers play a major role in drug delivery in vitro and in vivo. We report the bindings of biogenic polyamines, spermine (spm), and spermidine (spmd), and their synthetic analogues, 3,7,11,15-tetrazaheptadecane4HCl (BE-333) and 3,7,11,15,19-pentazahenicosane5HCl (BE-3333) with poly(ethylene glycol) PEG-3000, PEG-8000 and methoxy poly(ethylene glycol) anthracene (PEG-anthracene). Fourier transform infrared (FTIR), UV–visible and fluorescence spectroscopic were used to analyze polyamine binding mode, the binding constant and the effect of PEG compositions on polyamine–polymer interaction. Structural analysis showed that polyamines bind PEG through hydrophobic and hydrophilic contacts with overall binding constants of Kspm-PEG-3000 = 3.1 104 M1, Kspmd-PEG-3000 = 5.5 104 M1, KBE-333-PEG-3000 = 2.5 104 M1, KBE-3333-PEG-3000 = 1.5 105 M1, Kspm-PEG-8000 = 4.1105 M1, Kspmd-PEG-8000 = 7.5 105 M1, KBE-333-PEG-8000 = 4.5 104 M1, KBE-3333-PEG-8000 = 2.2 105 M1, Kspm-mPEG-ant = 6.5 105 M1, Kspmd-mPEG-ant = 1.1 106 M1, KBE-333-mPEG-ant = 2.2 105 M1 and KBE-3333-mPEG-ant = 6.9 104 M1. The number of binding sites (n) occupied by polyamines were from 0.2 to 0.5. Biogenic polyamines showed stronger affinity toward polymer complexation than synthetic polyamines, while weaker interaction was observed as polyamine cationic charges increased. Our results suggest that PEG and its derivative can act as carriers for delivering antitumor polyamine analogues to target tissues. Ó 2013 Elsevier B.V. All rights reserved.
1. Introduction Polymeric carriers, some of which physically encapsulate molecules of interest such as drug, gene and protein play an important role in modern pharmaceutical Technology. Among synthetic polymers, poly(ethylene) glycol and its derivatives show potential applications in gene and drug delivery due to their solubility, nontoxicity and biocompatibility [1,2]. Poly(ethylene glycol) is the most commonly used as non-ionic hydrophilic polymer with stealth behavior. Furthermore, PEG reduces the tendency of particles to aggregate by steric stabilization, thereby producing formulations with increased stability during storage and application [1]. PEGylation of synthetic polymers such as dendrimers is shown to reduce toxicity and increase biocompatibility [3–5]. Similarly, the effect of PEGylation on the toxicity and permeability of natural polymers such as chitosan and hydrogel has been recently reported [6,7]. The effect of PEG and its derivative on protein structure and function is well investigated [8–10]. Even though, the interactions of PEG and its derivatives with drugs are known [1,2], detailed structural analysis of PEG and mPEG with polyamines is not investigated. Therefore, it was of interest to study ⇑ Corresponding author. Tel.: +1 819 376 5011x3310; fax: +1 819 376 5084. E-mail address: [email protected] (H.A. Tajmir-Riahi). 1011-1344/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jphotobiol.2013.10.014
the interaction of PEG and PEG-anthracene nanoparticles with biogenic and synthetic polyamines, using different spectroscopic methods in order to evaluate the efficacy of PEG nanoparticles in drug delivery systems. Biogenic polyamines (Scheme 1) are essential for cell growth and differentiation, while polyamine analogues exert antitumor activity in multiple experimental model systems, including breast and lung cancer [11–15]. Synthetic polyamines (Scheme 1) can mimic some of the self-regulatory functions of biogenic polyamines but are unable to substitute for natural polyamines in their growth promoting role [16–23]. Natural polyamines are ubiquitous cellular cations and are involved in cell growth and differentiation [17]. They are capable of modulating gene expression and enzyme activities, activation of DNA synthesis, and facilitating protein– DNA interactions [23–30]. Even though interactions of biogenic and synthetic polyamines with biopolymers such as DNA and RNA and protein are well characterized [31–35], little is known about their interactions with synthetic polymers, such as PEG and its derivatives. In this report, we present the spectroscopic results on the binding of biogenic and synthetic polyamines with PEG-3000, PEG8000 and mPEG-anthracene, in aqueous solution, using a constant polymer concentration and different polyamine concentrations. Structural data regarding polyamine binding modes and the
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2. Experimental 2.1. Materials PEG-3000, PEG-8000, spermine4HCl and spermidine3HCl were purchased from Sigma Chemical Company and used as supplied. mPEG–anthracene was from Polymer Source (Quebec). Polyamine analogues, BE-333 and BE-3333, were synthesized in the laboratory of Dr. Akira Shirahata (Josai University, Saitama, Japan). Other chemicals were of reagent grade and used without further purification.
Scheme 1. Structures of biogenic and synthetic polyamines.
stability of polyamine–PEG complexes are presented and the possibility of delivery of polyamine analogues as antitumor drugs by PEG and its derivative is discussed here.
2.2. Preparation of stock solutions PEG and mPEG–anthracene solution (0.25 mM) were prepared in distilled water and diluted to various concentrations in Tris– HCl buffer. Polyamine solutions (0.25 mM) were prepared in water
Fig. 1. FTIR spectra and difference spectra (diff.) in the region of 1800–600 cm1 of hydrated films (pH 7.4) for free PEG-3000 (A), PEG-8000 (B) and mPEG-anthracene (C) and their complexes with spermine obtained at different spermine concentrations (indicated on the figure).
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Fig. 2. FTIR spectra and difference spectra (diff.) in the region of 1800–600 cm1 of hydrated films (pH 7.4) for free PEG-3000 (A), PEG-8000 (B) and mPEG-anthracene (C) and their complexes with spermidine obtained at different spermidine concentrations (indicated on the figure).
and diluted in Tris–HCl buffer. The pH of stock solutions was kept at 7 ± 0.2.
show no spectral changes (intensity or shifting) upon polyamine– PEG complex formation, and cancelled on spectral subtraction.
2.3. FTIR spectroscopic measurements
2.4. Fluorescence spectroscopy
Infrared spectra were recorded on a FTIR spectrometer (Impact 420 model), equipped with deuterated triglycine sulphate (DTGS) detector and KBr beam splitter, using AgBr windows. Polyamine solutions were added drop-wise to PEG solutions, with constant stirring to ensure the formation of homogeneous solutions and to reach target polyamine concentrations of 15, 30, and 60 lM and a final polymer concentration of 60 lM. Spectra were collected after 2 h incubation of polyamine and polymer solution at room temperature, using hydrated films. Interferograms were accumulated over the spectral range of 4000–600 cm1, with a nominal resolution of 4 cm1 and 100 scans. The difference spectra [(PEG solution + polyamine solution) – (PEG solution)] were generated, using PEG bands at 841 (PEG-3000), 842 (PEG-8000) and 819 cm1 (mPEG–anthracene). These vibrations are related to the polymers CAC stretching and ring skeletal modes [36,37] that
Fluorimetric experiments were carried out on a Perkin–Elmer LS55 Spectrometer. Stock solution of polymer (200 lM) was prepared at room temperature in Tris–HCl buffer (24 ± 1 °C). Various solutions of polyamine (1–100 lM) were prepared from the above stock solutions by successive dilutions. Stock solution of mPEG– anthracene (30 lM) in Tris–HCl buffer was also prepared at 24 ± 1 °C. Samples containing 0.06 ml of the above protein solution and various polymer solutions were mixed to obtain final polymer concentrations ranging from 1 to 100 lM with constant mPEG– anthracene (30 lM). The fluorescence spectra were recorded at kex = 330–350 nm and kem 390–450 nm related to anthracene fluoropore [38,39]. The intensity at 405 nm was used to calculate the binding constant (K) for polyamine–mPEG-anthracene– adducts was used to calculate the binding constant (K) according to previous reports [40–42].
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Fig. 3. FTIR spectra and difference spectra (diff.) in the region of 1800–600 cm1 of hydrated films (pH 7.4) for free PEG-3000 (A), PEG-8000 (B) and mPEG-anthracene (C) and their complexes with BE-333 obtained at different BE-333 concentrations (indicated on the figure).
On the assumption that there are (n) substantive binding sites for quencher (Q) on polymer (B), the quenching reaction can be shown as follows:
nQ þ B () Q n B
ð1Þ
log½ðF 0 FÞ=F ¼ log K A þ n log½Q
The binding constant (KA), can be calculated as: n
K A ¼ ½Q n B=½Q ½B
ð6Þ
ð2Þ
where [Q] and [B] are the quencher and polymer concentration, respectively, [QnB] is the concentration of non fluorescent fluorophore–quencher complex and [B0] gives total polymer concentration:
½Q n B ¼ ½B0 ½B
ð3Þ
K A ¼ ð½B0 ½BÞ=½Q n ½B
ð4Þ
The fluorescence intensity is proportional to the polymer concentration as described:
½B=½B0 / F=F 0
Results from fluorescence measurements can be used to estimate the binding constant of polymer–polyamine complex. From Eq. (4):
ð5Þ
The accessible fluorophore fraction (f) can be calculated by modified Stern–Volmer equation:
F 0 =ðF 0 FÞ ¼ 1=fK½Q þ 1=f
ð7Þ
where F0 is the initial fluorescence intensity and F is the fluorescence intensities in the presence of quenching agent (or interacting molecule). K is the Stern–Volmer quenching constant, [Q] is the molar concentration of quencher and f is the fraction of accessible fluorophore to a polar quencher, which indicates the fractional fluorescence contribution of the total emission for an interaction with a hydrophobic quencher [40–42]. The K will be calculated from F0/F = K[Q] + 1.
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Fig. 4. FTIR spectra and difference spectra (diff.) in the region of 1800–600 cm1 of hydrated films (pH 7.4) for free PEG-3000 (A), PEG-8000 (B) and mPEG-anthracene (C) and their complexes with BE-3333 obtained at different BE-3333 concentrations (indicated on the figure).
2.5. UV–visible spectroscopy The UV–visible spectra were recorded on a Perkin–Elmer Lambda spectrophotometer with a slit of 2 nm and scan speed of 400 nm min1. Quartz cuvettes of 1 cm were used. The absorbance measurements were performed at pH 7.0 by keeping the concentration of PEG constant (200 lM), while increasing polyamine concentrations (5–100 lM). The binding constants were obtained according to the method described by Connors [43]. It is assumed that the interaction between the ligand L and the substrate S is 1:1; for this reason a single complex SL (1:1) is formed. It was also assumed that the sites (and all the binding sites) are independent and all species obeyed the Beer’s law. A wavelength is selected at which the molar absorptivities eS (molar absorptivity of the substrate) and e11 (molar absorptivity of the complex) are different. In the absence of ligands and light path length (b) of 1 cm and at total substrate concentration St, the solution absorbance is given by the following equation:
Ao ¼ eS bSt
ð8Þ
At total concentration Lt of a ligand, the absorbance of a solution containing the same total substrate concentration is:
AL ¼ eS b½S þ eL b½L þ e11 b½SL
ð9Þ
where [S] is the concentration of the uncomplexed substrate, [L] the concentration of the uncomplexed ligand and [SL] is the concentration of the complex) which, combined with the mass balance on S and L, gives
AL ¼ eS bSt þ eL bLt þ De11 b½SL
ð10Þ
where De11 = e11 eS eL (eL molar absorptivity of the ligand). By measuring the solution absorbance against a reference containing ligand at the same total concentration Lt, the measured absorbance becomes
A ¼ eS bSt þ De11 b½SL
ð11Þ
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Fig. 5. FTIR spectra in the region of 3300–2800 cm1 of hydrated films (pH 7.4) for free PEG-3000 (A), PEG-8000 (B) and mPEG-anthracene (C) and their polyamine complexes obtained with 0.60 lM polymer and polyamine concentrations.
Combining Eq. (4) with the stability constant definition K11 = [SL]/[S][L], gives
DA ¼ K 11 De11 b½S½L
ð12Þ
where DA = A Ao. From the mass balance expression St = [S] + [SL] we get [S] = St/(1 + K11[L]), which is Eq. (5), giving Eq. (6) at the relationship between the observed absorbance change per centimeter and the system variables and parameters.
DA St K 11 De11 ½L ¼ b 1 þ K 11 ½L
ð13Þ
Eq. (6) is the binding isotherm, which shows the hyperbolic dependence on free ligand concentration. The double-reciprocal form of plotting the rectangular hyperbola 1y ¼ df 1x þ de, is based on the linearization of Eq. (6) according to the following equation,
b 1 1 þ ¼ DA St K 11 De11 ½L St De11
ð14Þ
Thus the double reciprocal plot of 1/DA versus 1/[L] is linear and the binding constant can be estimated from the following equation
K 11 ¼
intercept slope
ð15Þ
3. Results and discussion 3.1. FTIR spectral analysis of polyamine–PEG complexes Figs. 1–4 show the infrared spectra and difference spectra of PEG complexes with biogenic and synthetic polyamines. Spectral shifting was observed for the polymer C@O and CAO stretching and OH bending modes [36,37] due to polyamine hydrophilic interactions with PEG polar groups. The major infrared bands at 1633– 1629 cm1 (C@O stretch), 1297–1295 cm1 (CAO), 1057–1058 and 1038–1037, 1061 and 1039 cm1 (CAO and CAC stretch), in the infrared spectra of the free PEG-3000, PEG-8000 and mPEG-– anthracene exhibited shifting and intensity increases upon sperm-
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Fig. 6. Fluorescence emission spectra of mPEG-anthracene–polyamine systems in 10 mM Tris–HCl buffer pH 7.4 at 25 °C for A) polymer–DNA: (a) free mPEG-anthracene (30 lM), (b–k) with polymer–RNA complexes at 5, 10, 15, 20, 30, 40, 60, 80 and 100 lM with (l) free tRNA 100 lM. The plot of F0/(F0 F) as a function of 1/tRNA concentration. The binding constant K being the ratio of the intercept and the slope for (A0 ) mPEG-anthracene–tRNA.
Table 1 Binding parameters for polyamine–PEG complexes.
a b
Complexes
K (M1)a PEG-3000
K (M1) a PEG-8000
K (M1) b PEG-ant
kq (M1 S1)
n
Spm Spmd BE-333 BE-3333
3.1 104 5.5 104 2.5 104 1.5 104
4.1 105 7.5 105 4.5 104 2.2 104
6.5 105 1.1 106 2.2 105 6.9 104
1.3 1014 2.2 1014 4.4 1013 1.4 1013
0.2 0.2 0.3 0.5
From UV. From fluorescence spectroscopic measurements.
ine, spermidine, BE-333 and BE-3333 complexation (Figs. 1–4A–C). The observed spectral shifting was accompanied by gradual increase in intensity of the above vibrational frequencies in the difference spectra [(polymer solution + polyamine solution) – (polymer solution)] of polyamine–polymer complexes as polyamine concentration increased (Figs. 1–4A–C, diffs). The spectral
changes observed are attributed to the hydrophilic interactions of polyamine polar groups with PEG, OH, CAO and C@O groups. The hydrophilic interaction is more pronounced at high polyamine concentrations as evidenced by an increase in the intensity of several positive bands, centered at 1650–1000 cm1 in the difference spectra of polyamine–PEG complexes (Figs. 1–4A–C diffs 15 lM and 60 lM). 3.2. Hydrophobic and hydrophilic contacts The effect of polyamine–polymer complex formation on PEG antisymmetric and symmetric CH2 stretching vibrations in the region of 3000–2800 cm1 was investigated by infrared spectroscopy [37,38]. From Fig. 5A–C, the antisymmetric and symmetric CH2 bands of the free PEG-3000 at 3409, 2996, 2945 cm1 (Fig. 5A); free PEG-8000 at 3954, 2996, 2914 cm1 (Fig. 5B) and free mPEGanthracene at 3054, 2996 and 2943 cm1 (Fig. 5C) exhibited major shifting in the spectra of polyamine–polymer complexes (Fig. 5A–
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C). The observed spectral shifting for polymer CH2 vibrations is indicative of some degree of hydrophobic interactions for polyamine–polymer complexes. This is due to the hydrophobic contacts via polyamine hydrophobic parts (aliphatic CH2 groups) and the hydrophobic sections in PEG and mPEG-anthracene. These spectral changes were more pronounced in the case of mPEGanthracene with a major hydrophobic residue attached (Fig. 5C). More evidence for hydrophilic contact also comes from major shifting of the polymer OH stretching vibrations at about 3400 cm1 (free OH) and 3190 cm1 (strongly H-bonded OH group). This is consistent with the major spectral changes observed in the region 1800–600 cm1 due to polyamine–PEG hydrophilic interactions discussed above (Figs. 1–4A–C). 3.3. Fluorescence spectra and stability of polyamine–mPEGanthracene adducts Since polyamines are not fluorophores, the titration of mPEGanthracene was done against various polyamine concentrations, using mPEG-anthracene excitation at 330–350 nm and emission at 400–450 nm [38,39]. When mPEG-anthracene interacts with polyamine, fluorescence may change depending on the impact of such interaction on the mPEG-anthracene conformation or via direct quenching effect. The decrease of fluorescence intensity of mPEG-anthracene has been monitored at 405 nm for mPEG-anthracence–polyamine systems. The plot of F0/(F0 F) vs 1/[polyamine] is shown in Fig. 6A–D. Assuming that the observed changes in fluorescence come from the interaction between mPEG-anthracene and polyamine, the quenching constant can be taken as the binding constant of the complex formation. The K value given here is average of the four-replicate and six-replicate runs for polyamine–mPEGanthracene systems, each run involving several different concentrations of polyamine (Fig. 6A–D). The binding constants obtained were Kspm-mPEG-ant = 6.5 105 M1, Kspmd-mPEG-ant = 1.1 106 M1, KBE-333-mPEG-ant = 2.2 105 M1 and KBE-3333-mPEG-ant = 6.9 104 M1 (Fig. 6A0 –D0 and Table 1). The association constants calculated for the polyamine–mPEG-anthracene adducts suggest strong affinity for polyamine–mPEG-anthracene interaction. The f values obtained 0.25–0.65 suggest that a major part of fluorophore was exposed to polyamine quenching as a result of hydrophobic interaction, which is consistent with our infrared spectroscopic results discussed above (hydrophobic contacts). The number of binding sites occupied by polyamines (n) is calculated from log [(F0 F)/F] = log KS + n log [polyamine] for the static quenching [41,42,44,45]. The linear plot of log [(F0 F]/F] as a function of log [polyamine] is shown in Fig. 7A–D. The n values from the slope of the straight line were 0.2–0.5 for polyamine– PEG complexes (Fig. 7 and Table 1). To verify the presence of static or dynamic quenching in polymer-b-LG complexes the quenching coefficient constant kq was calculated from plot of F0/F against Q. The plot of F0/F versus Q is straight line for polyamine–PEG adducts indicating that the quenching is mainly static in these polyamine–polymer complexes. The kq was estimated according to the Stern–Volmer equation:
F 0 =F ¼ 1 þ kq t0 ½Q ¼ 1 þ K sv ½Q
Fig. 7. The plot of Log (F0 F)/F as a function of Log (polyamine concentrations) spm-mPEG-Ant (A), spmd-mPEG-Ant (B), BE-333-mPEG-Ant (C) and BE-3333mPEG-Ant (D).
ð16Þ
where F0 and F are the fluorescence intensities in the absence and presence of quencher, [Q] is the quencher concentration and Ksv is the Stern–Volmer quenching constant [40], which can be written as Ksv = kqt0; where kq is the bimolecular quenching rate constant and t0 is the lifetime of the fluorophore in the absence of quencher about 5 ns for free anthracene [38]. The quenching constants (kq) were 1.3 1014 M1 s1 (spm-mPEG-anthracene), 2.2 1014 M1 s1 (spmd-mPEG-anthracene), 4.4 1013 M1 s1 (BE-333-mPEGanthacene) and 1.4 1013 M1 s1 (BE-3333-mPEG-anthracene)
Fig. 8. UV–visible spectra of PEG-3000 its complexes with free PEG, at 200 lM (a) and complexes (b–g) at 5, 10, 20, 40, 80 and 100 lM. Inset: plot of 1/(A0 A) vs (1/ polyamine concentration) and binding constant (K) for spm-PEG-3000.
(Table 1). Since these values are much greater than the maximum collisional quenching constant (2.0 1010 M1/s), thus static quenching is dominant in these polyamine–mPEG-anthracene complexes [46].
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3.4. UV–visible spectra and stability of polyamine–PEG complexes The UV spectra of polyamine–PEG complexes are presented in Fig. 8. There is clear evidence that polyamine–polymer complex formation occurred, as major decrease in the intensity of PEG absorption band 200–250 nm was observed [36,37]. The spectral changes are more pronounced in the case of biogenic polyamines than those of polyamine analogues (Fig. 8). The polyamine–PEG binding constants were calculated (according to the method described in Section 2 (using plots of 1/(A0 A) vs (1/polyamine concentrations) (Fig. 8). The calculated binding constants are: Kspm-PEG-3000 = 3.1 104 M1, Kspmd-PEG-3000 = 5.5 104 M1, KBE-333-PEG-3000 = 2.5 104 M1, KBE-3333-PEG-3000 = 1.5 105 M1, Kspm-PEG-8000 = 4.1 105 M1, Kspmd-PEG-8000 5 1 = 7.5 10 M , KBE-333-PEG-8000 = 4.5 104 M1, KBE-3333-PEG-8000 = 2.2 105 M1 (Table 1). The binding affinity of biogenic polyamines toward PEG was stronger than that of synthetic polyamines, while weaker interaction was observed as polyamine cationic charge increased (Table 1). The reason why biogenic polyamine–PEG are more stable than those of the synthetic polyamines can be due to other factors such as the primary amines ðANHþ 3 Þ in biogenic polyamines that possess a higher density of positive charge than the secondary ones ðANHþ 2 Þ, in synthetic polyamines [45] and also the presence of more hydrophobic contacts in the biogenic polyamine–polymer complexes. 4. Conclusions Biogenic and synthetic polyamines bind strongly to PEG and mPEG-anthracene with more stable complexes formed with biogenic polyamines. Both hydrophilic and hydrophobic contacts are observed in polyamine–PEG complexation. PEG and its derivatives can act as polyamine delivery vehicles, especially for polyamine analogues that are used as cancer chemotherapeutic agents. Abbreviations PEG mPEG-ant spm spmd BE-333 BE-3333 FTIR
poly(ethylene glycol) methoxy poly(ethylene glycol) anthracene spermine spermidine 3,7,11,15-tetrazaheptadecane4HCl 3,7,11,15,19-pentazahenicosane5HCl Fourier transform infrared spectroscopy
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N’soukpoé-Kossi, A. Ahmed Ouameur, T. Thomas, A. Shirahata, T.J. Thomas, H.A. Tajmir-Riahi, DNA interaction with antitumor polyamine analogues, Biomacromolecules 9 (2008) 2712–2718. [34] C.N. N’soukpoé-Kossi, A. Ahmed Ouameur, T. Thomas, T.J. Thomas, H.A. TajmirRiahi, Interaction tRNA with antitumor polyamine analogues, Biochem. Cell Biol. 87 (2009) 621–630.
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[41] E. Froehlich, J.S. Mandeville, D. Arnold, L. Kreplak, H.A. Tajmir-Riahi, PEG and mPEG-anthracene induce DNA condensation and particle formation, J. Phys. Chem. B 115 (2011) 9873–9879. [42] E. Froehlich, J.S. Mandeville, D. Arnold, L. Kreplak, H.A. Tajmir-Riahi, The effect of PEG and mPEG-anthracene on tRNA aggregation and particle formation, Biomacromolecules 13 (2012) 282–287. [43] K. Connors, Binding Constants: The Measurement of Molecular Complex Stability, John Wiley & Sons, New York, 1987. [44] S. Dubeau, P. Bourassa, T.J. Thomas, H.A. Tajmir-Riah, Biogenic and synthetic polyamines bind bovine serum albumin, Biomacromolecules 11 (2010) 1507– 1515. [45] J. Essemine, I. Hasni, R. Carpentier, T.J. Thomas, H.A. Tajmir-Riahi, Binding of biogenic and synthetic polyamines to beta-lactoglobulin, Int. J. Biol. Macromol. 49 (2011) 201–209. [46] G. Zhang, Q. Que, J. Pan, J. Guo, Study of the interaction between icariin and human serum albumin by fluorescence spectroscopy, J. Mol. Struct. 881 (2008) 132–138.
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Journal of Photochemistry and Photobiology B: Biology journal homepage: www.elsevier.com/locate/jphotobiol
Encapsulation of biogenic and synthetic polyamines by nanoparticles PEG and mPEG-anthracene S. Sanyakamdhorn, P. Chanphai, H.A. Tajmir-Riahi ⇑ Department of Chemistry–Physics, University of Québec in Trois-Rivières, C.P. 500, Trois-Rivières, Québec G9A 5H7, Canada
a r t i c l e
i n f o
Article history: Received 30 September 2013 Received in revised form 20 October 2013 Accepted 22 October 2013 Available online 1 November 2013 Keywords: PEG Polymer Polyamine Binding site FTIR Fluorescence spectroscopy
a b s t r a c t Synthetic polymers play a major role in drug delivery in vitro and in vivo. We report the bindings of biogenic polyamines, spermine (spm), and spermidine (spmd), and their synthetic analogues, 3,7,11,15-tetrazaheptadecane4HCl (BE-333) and 3,7,11,15,19-pentazahenicosane5HCl (BE-3333) with poly(ethylene glycol) PEG-3000, PEG-8000 and methoxy poly(ethylene glycol) anthracene (PEG-anthracene). Fourier transform infrared (FTIR), UV–visible and fluorescence spectroscopic were used to analyze polyamine binding mode, the binding constant and the effect of PEG compositions on polyamine–polymer interaction. Structural analysis showed that polyamines bind PEG through hydrophobic and hydrophilic contacts with overall binding constants of Kspm-PEG-3000 = 3.1 104 M1, Kspmd-PEG-3000 = 5.5 104 M1, KBE-333-PEG-3000 = 2.5 104 M1, KBE-3333-PEG-3000 = 1.5 105 M1, Kspm-PEG-8000 = 4.1105 M1, Kspmd-PEG-8000 = 7.5 105 M1, KBE-333-PEG-8000 = 4.5 104 M1, KBE-3333-PEG-8000 = 2.2 105 M1, Kspm-mPEG-ant = 6.5 105 M1, Kspmd-mPEG-ant = 1.1 106 M1, KBE-333-mPEG-ant = 2.2 105 M1 and KBE-3333-mPEG-ant = 6.9 104 M1. The number of binding sites (n) occupied by polyamines were from 0.2 to 0.5. Biogenic polyamines showed stronger affinity toward polymer complexation than synthetic polyamines, while weaker interaction was observed as polyamine cationic charges increased. Our results suggest that PEG and its derivative can act as carriers for delivering antitumor polyamine analogues to target tissues. Ó 2013 Elsevier B.V. All rights reserved.
1. Introduction Polymeric carriers, some of which physically encapsulate molecules of interest such as drug, gene and protein play an important role in modern pharmaceutical Technology. Among synthetic polymers, poly(ethylene) glycol and its derivatives show potential applications in gene and drug delivery due to their solubility, nontoxicity and biocompatibility [1,2]. Poly(ethylene glycol) is the most commonly used as non-ionic hydrophilic polymer with stealth behavior. Furthermore, PEG reduces the tendency of particles to aggregate by steric stabilization, thereby producing formulations with increased stability during storage and application [1]. PEGylation of synthetic polymers such as dendrimers is shown to reduce toxicity and increase biocompatibility [3–5]. Similarly, the effect of PEGylation on the toxicity and permeability of natural polymers such as chitosan and hydrogel has been recently reported [6,7]. The effect of PEG and its derivative on protein structure and function is well investigated [8–10]. Even though, the interactions of PEG and its derivatives with drugs are known [1,2], detailed structural analysis of PEG and mPEG with polyamines is not investigated. Therefore, it was of interest to study ⇑ Corresponding author. Tel.: +1 819 376 5011x3310; fax: +1 819 376 5084. E-mail address: [email protected] (H.A. Tajmir-Riahi). 1011-1344/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jphotobiol.2013.10.014
the interaction of PEG and PEG-anthracene nanoparticles with biogenic and synthetic polyamines, using different spectroscopic methods in order to evaluate the efficacy of PEG nanoparticles in drug delivery systems. Biogenic polyamines (Scheme 1) are essential for cell growth and differentiation, while polyamine analogues exert antitumor activity in multiple experimental model systems, including breast and lung cancer [11–15]. Synthetic polyamines (Scheme 1) can mimic some of the self-regulatory functions of biogenic polyamines but are unable to substitute for natural polyamines in their growth promoting role [16–23]. Natural polyamines are ubiquitous cellular cations and are involved in cell growth and differentiation [17]. They are capable of modulating gene expression and enzyme activities, activation of DNA synthesis, and facilitating protein– DNA interactions [23–30]. Even though interactions of biogenic and synthetic polyamines with biopolymers such as DNA and RNA and protein are well characterized [31–35], little is known about their interactions with synthetic polymers, such as PEG and its derivatives. In this report, we present the spectroscopic results on the binding of biogenic and synthetic polyamines with PEG-3000, PEG8000 and mPEG-anthracene, in aqueous solution, using a constant polymer concentration and different polyamine concentrations. Structural data regarding polyamine binding modes and the
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2. Experimental 2.1. Materials PEG-3000, PEG-8000, spermine4HCl and spermidine3HCl were purchased from Sigma Chemical Company and used as supplied. mPEG–anthracene was from Polymer Source (Quebec). Polyamine analogues, BE-333 and BE-3333, were synthesized in the laboratory of Dr. Akira Shirahata (Josai University, Saitama, Japan). Other chemicals were of reagent grade and used without further purification.
Scheme 1. Structures of biogenic and synthetic polyamines.
stability of polyamine–PEG complexes are presented and the possibility of delivery of polyamine analogues as antitumor drugs by PEG and its derivative is discussed here.
2.2. Preparation of stock solutions PEG and mPEG–anthracene solution (0.25 mM) were prepared in distilled water and diluted to various concentrations in Tris– HCl buffer. Polyamine solutions (0.25 mM) were prepared in water
Fig. 1. FTIR spectra and difference spectra (diff.) in the region of 1800–600 cm1 of hydrated films (pH 7.4) for free PEG-3000 (A), PEG-8000 (B) and mPEG-anthracene (C) and their complexes with spermine obtained at different spermine concentrations (indicated on the figure).
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Fig. 2. FTIR spectra and difference spectra (diff.) in the region of 1800–600 cm1 of hydrated films (pH 7.4) for free PEG-3000 (A), PEG-8000 (B) and mPEG-anthracene (C) and their complexes with spermidine obtained at different spermidine concentrations (indicated on the figure).
and diluted in Tris–HCl buffer. The pH of stock solutions was kept at 7 ± 0.2.
show no spectral changes (intensity or shifting) upon polyamine– PEG complex formation, and cancelled on spectral subtraction.
2.3. FTIR spectroscopic measurements
2.4. Fluorescence spectroscopy
Infrared spectra were recorded on a FTIR spectrometer (Impact 420 model), equipped with deuterated triglycine sulphate (DTGS) detector and KBr beam splitter, using AgBr windows. Polyamine solutions were added drop-wise to PEG solutions, with constant stirring to ensure the formation of homogeneous solutions and to reach target polyamine concentrations of 15, 30, and 60 lM and a final polymer concentration of 60 lM. Spectra were collected after 2 h incubation of polyamine and polymer solution at room temperature, using hydrated films. Interferograms were accumulated over the spectral range of 4000–600 cm1, with a nominal resolution of 4 cm1 and 100 scans. The difference spectra [(PEG solution + polyamine solution) – (PEG solution)] were generated, using PEG bands at 841 (PEG-3000), 842 (PEG-8000) and 819 cm1 (mPEG–anthracene). These vibrations are related to the polymers CAC stretching and ring skeletal modes [36,37] that
Fluorimetric experiments were carried out on a Perkin–Elmer LS55 Spectrometer. Stock solution of polymer (200 lM) was prepared at room temperature in Tris–HCl buffer (24 ± 1 °C). Various solutions of polyamine (1–100 lM) were prepared from the above stock solutions by successive dilutions. Stock solution of mPEG– anthracene (30 lM) in Tris–HCl buffer was also prepared at 24 ± 1 °C. Samples containing 0.06 ml of the above protein solution and various polymer solutions were mixed to obtain final polymer concentrations ranging from 1 to 100 lM with constant mPEG– anthracene (30 lM). The fluorescence spectra were recorded at kex = 330–350 nm and kem 390–450 nm related to anthracene fluoropore [38,39]. The intensity at 405 nm was used to calculate the binding constant (K) for polyamine–mPEG-anthracene– adducts was used to calculate the binding constant (K) according to previous reports [40–42].
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Fig. 3. FTIR spectra and difference spectra (diff.) in the region of 1800–600 cm1 of hydrated films (pH 7.4) for free PEG-3000 (A), PEG-8000 (B) and mPEG-anthracene (C) and their complexes with BE-333 obtained at different BE-333 concentrations (indicated on the figure).
On the assumption that there are (n) substantive binding sites for quencher (Q) on polymer (B), the quenching reaction can be shown as follows:
nQ þ B () Q n B
ð1Þ
log½ðF 0 FÞ=F ¼ log K A þ n log½Q
The binding constant (KA), can be calculated as: n
K A ¼ ½Q n B=½Q ½B
ð6Þ
ð2Þ
where [Q] and [B] are the quencher and polymer concentration, respectively, [QnB] is the concentration of non fluorescent fluorophore–quencher complex and [B0] gives total polymer concentration:
½Q n B ¼ ½B0 ½B
ð3Þ
K A ¼ ð½B0 ½BÞ=½Q n ½B
ð4Þ
The fluorescence intensity is proportional to the polymer concentration as described:
½B=½B0 / F=F 0
Results from fluorescence measurements can be used to estimate the binding constant of polymer–polyamine complex. From Eq. (4):
ð5Þ
The accessible fluorophore fraction (f) can be calculated by modified Stern–Volmer equation:
F 0 =ðF 0 FÞ ¼ 1=fK½Q þ 1=f
ð7Þ
where F0 is the initial fluorescence intensity and F is the fluorescence intensities in the presence of quenching agent (or interacting molecule). K is the Stern–Volmer quenching constant, [Q] is the molar concentration of quencher and f is the fraction of accessible fluorophore to a polar quencher, which indicates the fractional fluorescence contribution of the total emission for an interaction with a hydrophobic quencher [40–42]. The K will be calculated from F0/F = K[Q] + 1.
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Fig. 4. FTIR spectra and difference spectra (diff.) in the region of 1800–600 cm1 of hydrated films (pH 7.4) for free PEG-3000 (A), PEG-8000 (B) and mPEG-anthracene (C) and their complexes with BE-3333 obtained at different BE-3333 concentrations (indicated on the figure).
2.5. UV–visible spectroscopy The UV–visible spectra were recorded on a Perkin–Elmer Lambda spectrophotometer with a slit of 2 nm and scan speed of 400 nm min1. Quartz cuvettes of 1 cm were used. The absorbance measurements were performed at pH 7.0 by keeping the concentration of PEG constant (200 lM), while increasing polyamine concentrations (5–100 lM). The binding constants were obtained according to the method described by Connors [43]. It is assumed that the interaction between the ligand L and the substrate S is 1:1; for this reason a single complex SL (1:1) is formed. It was also assumed that the sites (and all the binding sites) are independent and all species obeyed the Beer’s law. A wavelength is selected at which the molar absorptivities eS (molar absorptivity of the substrate) and e11 (molar absorptivity of the complex) are different. In the absence of ligands and light path length (b) of 1 cm and at total substrate concentration St, the solution absorbance is given by the following equation:
Ao ¼ eS bSt
ð8Þ
At total concentration Lt of a ligand, the absorbance of a solution containing the same total substrate concentration is:
AL ¼ eS b½S þ eL b½L þ e11 b½SL
ð9Þ
where [S] is the concentration of the uncomplexed substrate, [L] the concentration of the uncomplexed ligand and [SL] is the concentration of the complex) which, combined with the mass balance on S and L, gives
AL ¼ eS bSt þ eL bLt þ De11 b½SL
ð10Þ
where De11 = e11 eS eL (eL molar absorptivity of the ligand). By measuring the solution absorbance against a reference containing ligand at the same total concentration Lt, the measured absorbance becomes
A ¼ eS bSt þ De11 b½SL
ð11Þ
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Fig. 5. FTIR spectra in the region of 3300–2800 cm1 of hydrated films (pH 7.4) for free PEG-3000 (A), PEG-8000 (B) and mPEG-anthracene (C) and their polyamine complexes obtained with 0.60 lM polymer and polyamine concentrations.
Combining Eq. (4) with the stability constant definition K11 = [SL]/[S][L], gives
DA ¼ K 11 De11 b½S½L
ð12Þ
where DA = A Ao. From the mass balance expression St = [S] + [SL] we get [S] = St/(1 + K11[L]), which is Eq. (5), giving Eq. (6) at the relationship between the observed absorbance change per centimeter and the system variables and parameters.
DA St K 11 De11 ½L ¼ b 1 þ K 11 ½L
ð13Þ
Eq. (6) is the binding isotherm, which shows the hyperbolic dependence on free ligand concentration. The double-reciprocal form of plotting the rectangular hyperbola 1y ¼ df 1x þ de, is based on the linearization of Eq. (6) according to the following equation,
b 1 1 þ ¼ DA St K 11 De11 ½L St De11
ð14Þ
Thus the double reciprocal plot of 1/DA versus 1/[L] is linear and the binding constant can be estimated from the following equation
K 11 ¼
intercept slope
ð15Þ
3. Results and discussion 3.1. FTIR spectral analysis of polyamine–PEG complexes Figs. 1–4 show the infrared spectra and difference spectra of PEG complexes with biogenic and synthetic polyamines. Spectral shifting was observed for the polymer C@O and CAO stretching and OH bending modes [36,37] due to polyamine hydrophilic interactions with PEG polar groups. The major infrared bands at 1633– 1629 cm1 (C@O stretch), 1297–1295 cm1 (CAO), 1057–1058 and 1038–1037, 1061 and 1039 cm1 (CAO and CAC stretch), in the infrared spectra of the free PEG-3000, PEG-8000 and mPEG-– anthracene exhibited shifting and intensity increases upon sperm-
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Fig. 6. Fluorescence emission spectra of mPEG-anthracene–polyamine systems in 10 mM Tris–HCl buffer pH 7.4 at 25 °C for A) polymer–DNA: (a) free mPEG-anthracene (30 lM), (b–k) with polymer–RNA complexes at 5, 10, 15, 20, 30, 40, 60, 80 and 100 lM with (l) free tRNA 100 lM. The plot of F0/(F0 F) as a function of 1/tRNA concentration. The binding constant K being the ratio of the intercept and the slope for (A0 ) mPEG-anthracene–tRNA.
Table 1 Binding parameters for polyamine–PEG complexes.
a b
Complexes
K (M1)a PEG-3000
K (M1) a PEG-8000
K (M1) b PEG-ant
kq (M1 S1)
n
Spm Spmd BE-333 BE-3333
3.1 104 5.5 104 2.5 104 1.5 104
4.1 105 7.5 105 4.5 104 2.2 104
6.5 105 1.1 106 2.2 105 6.9 104
1.3 1014 2.2 1014 4.4 1013 1.4 1013
0.2 0.2 0.3 0.5
From UV. From fluorescence spectroscopic measurements.
ine, spermidine, BE-333 and BE-3333 complexation (Figs. 1–4A–C). The observed spectral shifting was accompanied by gradual increase in intensity of the above vibrational frequencies in the difference spectra [(polymer solution + polyamine solution) – (polymer solution)] of polyamine–polymer complexes as polyamine concentration increased (Figs. 1–4A–C, diffs). The spectral
changes observed are attributed to the hydrophilic interactions of polyamine polar groups with PEG, OH, CAO and C@O groups. The hydrophilic interaction is more pronounced at high polyamine concentrations as evidenced by an increase in the intensity of several positive bands, centered at 1650–1000 cm1 in the difference spectra of polyamine–PEG complexes (Figs. 1–4A–C diffs 15 lM and 60 lM). 3.2. Hydrophobic and hydrophilic contacts The effect of polyamine–polymer complex formation on PEG antisymmetric and symmetric CH2 stretching vibrations in the region of 3000–2800 cm1 was investigated by infrared spectroscopy [37,38]. From Fig. 5A–C, the antisymmetric and symmetric CH2 bands of the free PEG-3000 at 3409, 2996, 2945 cm1 (Fig. 5A); free PEG-8000 at 3954, 2996, 2914 cm1 (Fig. 5B) and free mPEGanthracene at 3054, 2996 and 2943 cm1 (Fig. 5C) exhibited major shifting in the spectra of polyamine–polymer complexes (Fig. 5A–
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C). The observed spectral shifting for polymer CH2 vibrations is indicative of some degree of hydrophobic interactions for polyamine–polymer complexes. This is due to the hydrophobic contacts via polyamine hydrophobic parts (aliphatic CH2 groups) and the hydrophobic sections in PEG and mPEG-anthracene. These spectral changes were more pronounced in the case of mPEGanthracene with a major hydrophobic residue attached (Fig. 5C). More evidence for hydrophilic contact also comes from major shifting of the polymer OH stretching vibrations at about 3400 cm1 (free OH) and 3190 cm1 (strongly H-bonded OH group). This is consistent with the major spectral changes observed in the region 1800–600 cm1 due to polyamine–PEG hydrophilic interactions discussed above (Figs. 1–4A–C). 3.3. Fluorescence spectra and stability of polyamine–mPEGanthracene adducts Since polyamines are not fluorophores, the titration of mPEGanthracene was done against various polyamine concentrations, using mPEG-anthracene excitation at 330–350 nm and emission at 400–450 nm [38,39]. When mPEG-anthracene interacts with polyamine, fluorescence may change depending on the impact of such interaction on the mPEG-anthracene conformation or via direct quenching effect. The decrease of fluorescence intensity of mPEG-anthracene has been monitored at 405 nm for mPEG-anthracence–polyamine systems. The plot of F0/(F0 F) vs 1/[polyamine] is shown in Fig. 6A–D. Assuming that the observed changes in fluorescence come from the interaction between mPEG-anthracene and polyamine, the quenching constant can be taken as the binding constant of the complex formation. The K value given here is average of the four-replicate and six-replicate runs for polyamine–mPEGanthracene systems, each run involving several different concentrations of polyamine (Fig. 6A–D). The binding constants obtained were Kspm-mPEG-ant = 6.5 105 M1, Kspmd-mPEG-ant = 1.1 106 M1, KBE-333-mPEG-ant = 2.2 105 M1 and KBE-3333-mPEG-ant = 6.9 104 M1 (Fig. 6A0 –D0 and Table 1). The association constants calculated for the polyamine–mPEG-anthracene adducts suggest strong affinity for polyamine–mPEG-anthracene interaction. The f values obtained 0.25–0.65 suggest that a major part of fluorophore was exposed to polyamine quenching as a result of hydrophobic interaction, which is consistent with our infrared spectroscopic results discussed above (hydrophobic contacts). The number of binding sites occupied by polyamines (n) is calculated from log [(F0 F)/F] = log KS + n log [polyamine] for the static quenching [41,42,44,45]. The linear plot of log [(F0 F]/F] as a function of log [polyamine] is shown in Fig. 7A–D. The n values from the slope of the straight line were 0.2–0.5 for polyamine– PEG complexes (Fig. 7 and Table 1). To verify the presence of static or dynamic quenching in polymer-b-LG complexes the quenching coefficient constant kq was calculated from plot of F0/F against Q. The plot of F0/F versus Q is straight line for polyamine–PEG adducts indicating that the quenching is mainly static in these polyamine–polymer complexes. The kq was estimated according to the Stern–Volmer equation:
F 0 =F ¼ 1 þ kq t0 ½Q ¼ 1 þ K sv ½Q
Fig. 7. The plot of Log (F0 F)/F as a function of Log (polyamine concentrations) spm-mPEG-Ant (A), spmd-mPEG-Ant (B), BE-333-mPEG-Ant (C) and BE-3333mPEG-Ant (D).
ð16Þ
where F0 and F are the fluorescence intensities in the absence and presence of quencher, [Q] is the quencher concentration and Ksv is the Stern–Volmer quenching constant [40], which can be written as Ksv = kqt0; where kq is the bimolecular quenching rate constant and t0 is the lifetime of the fluorophore in the absence of quencher about 5 ns for free anthracene [38]. The quenching constants (kq) were 1.3 1014 M1 s1 (spm-mPEG-anthracene), 2.2 1014 M1 s1 (spmd-mPEG-anthracene), 4.4 1013 M1 s1 (BE-333-mPEGanthacene) and 1.4 1013 M1 s1 (BE-3333-mPEG-anthracene)
Fig. 8. UV–visible spectra of PEG-3000 its complexes with free PEG, at 200 lM (a) and complexes (b–g) at 5, 10, 20, 40, 80 and 100 lM. Inset: plot of 1/(A0 A) vs (1/ polyamine concentration) and binding constant (K) for spm-PEG-3000.
(Table 1). Since these values are much greater than the maximum collisional quenching constant (2.0 1010 M1/s), thus static quenching is dominant in these polyamine–mPEG-anthracene complexes [46].
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3.4. UV–visible spectra and stability of polyamine–PEG complexes The UV spectra of polyamine–PEG complexes are presented in Fig. 8. There is clear evidence that polyamine–polymer complex formation occurred, as major decrease in the intensity of PEG absorption band 200–250 nm was observed [36,37]. The spectral changes are more pronounced in the case of biogenic polyamines than those of polyamine analogues (Fig. 8). The polyamine–PEG binding constants were calculated (according to the method described in Section 2 (using plots of 1/(A0 A) vs (1/polyamine concentrations) (Fig. 8). The calculated binding constants are: Kspm-PEG-3000 = 3.1 104 M1, Kspmd-PEG-3000 = 5.5 104 M1, KBE-333-PEG-3000 = 2.5 104 M1, KBE-3333-PEG-3000 = 1.5 105 M1, Kspm-PEG-8000 = 4.1 105 M1, Kspmd-PEG-8000 5 1 = 7.5 10 M , KBE-333-PEG-8000 = 4.5 104 M1, KBE-3333-PEG-8000 = 2.2 105 M1 (Table 1). The binding affinity of biogenic polyamines toward PEG was stronger than that of synthetic polyamines, while weaker interaction was observed as polyamine cationic charge increased (Table 1). The reason why biogenic polyamine–PEG are more stable than those of the synthetic polyamines can be due to other factors such as the primary amines ðANHþ 3 Þ in biogenic polyamines that possess a higher density of positive charge than the secondary ones ðANHþ 2 Þ, in synthetic polyamines [45] and also the presence of more hydrophobic contacts in the biogenic polyamine–polymer complexes. 4. Conclusions Biogenic and synthetic polyamines bind strongly to PEG and mPEG-anthracene with more stable complexes formed with biogenic polyamines. Both hydrophilic and hydrophobic contacts are observed in polyamine–PEG complexation. PEG and its derivatives can act as polyamine delivery vehicles, especially for polyamine analogues that are used as cancer chemotherapeutic agents. Abbreviations PEG mPEG-ant spm spmd BE-333 BE-3333 FTIR
poly(ethylene glycol) methoxy poly(ethylene glycol) anthracene spermine spermidine 3,7,11,15-tetrazaheptadecane4HCl 3,7,11,15,19-pentazahenicosane5HCl Fourier transform infrared spectroscopy
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