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Enhanced Drug Delivery into Cell Cytosol via Glycoprotein H-Derived Peptide Conjugated Nanoemulsions Teresa Fotticchia, Raffaele Vecchione, Pasqualina Liana Scognamiglio, Daniela Guarnieri, Vincenzo Calcagno, Concetta Di Natale, Chiara Attanasio, Maria De Gregorio, Chiara Di Cicco, Vincenzo Quagliariello, Nicola Maurea, Antonio Barbieri, Luca Raiola, Rosario Vincenzo Iaffaioli, and Paolo Antonio Netti ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.7b03058 • Publication Date (Web): 18 Aug 2017 Downloaded from http://pubs.acs.org on August 19, 2017
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Enhanced Drug Delivery into Cell Cytosol via Glycoprotein H-Derived Peptide Conjugated Nanoemulsions Teresa Fotticchiaa,+, Raffaele Vecchionea,b,+,*, Pasqualina Liana Scognamiglioa, Daniela Guarnieria, Vincenzo Calcagnoa, Concetta Di Natalea, Chiara Attanasioa, Maria De Gregorioa, Chiara Di Ciccoa, Vincenzo Quagliarielloc, Nicola Mauread, Antonio Barbierif, Luca Raiolaa, Rosario V. Iaffaiolic, Paolo A. Nettia,b a
Center for Advanced Biomaterials for Health Care@CRIB, Istituto Italiano di Tecnologia, Largo Barsanti e Matteucci 53, Napoli 80125, Italy b
Interdisciplinary Research Center of Biomaterials, CRIB, University Federico II, P.le Tecchio, 80 80125, Naples, Italy
c
Medical Oncology, Abdominal Department, National Cancer Institute G. Pascale Foundation, Napoli, 80131, Italy. d
Division of Cardiology, National Cancer Institute G. Pascale Foundation, Napoli, 80131, Italy.
f
Animal Facility Unit, Department of Research, National Cancer Institute G. Pascale Foundation, Napoli, 80131, Italy Dr. Raffaele Vecchione:
[email protected]; tel:+3908119933127; Fax: +3908119933140
ABSTRACT The key role of nanocarriers in improving the pharmacological properties of commonly used drugs is recognized worldwide. It is also known that in the development of new effective nanocarriers the use of targeting moieties integrated on their surface is essential. Herein, we propose a nanocarrier based on an oil in water nanoemulsion coated with a membranotropic peptide derived from the glycoprotein H of Herpes simplex virus 1, known as gH625, in order to reduce endo-lysosomal accumulation and to enhance cytosolic localization. In addition, we showed an enhanced antiinflammatory activity of curcumin, a bioactive compound isolated from Curcuma longa plant, when loaded into our engineered nanocarriers. This effect is a consequence of a higher uptake combined with a high curcumin preservation exerted by the active nanocapsules compared to control ones. When loaded into our nanocapsules, indeed, curcumin molecules are directly internalized into the cytosol rather than into lysosomes. Further, in order to extend the in vitro experimental setting with a more complex model and to explore the possibility to use our nanocarriers for further biological applications, we tested their performance in a 3D sprouting angiogenesis model. Finally, we showed ACS Paragon Plus Environment
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promising preliminary in vivo results by assessing the anti-inflammatory properties of the proposed nanocarrier. KEYWORDS: nanoemulsions, nanocapsules, curcumin, gH625, drug delivery Oil in water nanoemulsions (O/W NEs) are an ideal vehicle for drug delivery thanks to their ability to dissolve large quantities of hydrophobic drugs as well as contrasting agents.1-6 There is currently a great deal of research in this field, especially because of the poor water solubility of many newly discovered drugs7, including natural drugs such as nutraceuticals which are mainly lipophilic.8-11 In addition, O/W NEs are able to protect drugs from hydrolysis and enzymatic degradation under physiological conditions and to be properly stabilized as previously reported.8,
12-14
These basic
properties make O/W NEs promising drug delivery systems, so that a number of pharmaceutical formulations based on them are already on the market while several others are under preclinical and clinical stage of development.15 Further, it was shown that O/W NEs are able to actively target cells or tissues when specific moieties coat their surface.7 In this work, we developed O/W NEs which mostly accumulate into cytosol rather than into lysosomes thanks to the conjugation to a membranotropic peptide through supramolecular nanoassembly. This nineteen-residues peptide, identified as gH625, is derived from the glycoprotein H (gH) of Herpes simplex virus 1 and belongs to a class of hydrophobic peptides able to induce a temporary and local membrane destabilization with following reorganization.16-18 This is the consequence of an efficient interaction between the peptide and the biological membranes that promotes lipid membrane-reorganizing processes, such as fusion or pore formation.17-19 We recently demonstrated that surface decoration of rigid polymer nanoparticles with gH625 enables their entrance into the cells through a non-conventional path and their crossing through a confluent layer of mouse microvascular endothelial cells (bEnd.3) both in static and in dynamic conditions.19-21 In this work we show that although the described extralysosomal path is a peculiar mechanism of entrance of nanoparticles into the cells, it can easily be translated to soft matter nanocarriers such as O/W NEs. Herein, a ternary poly-L-lysinestreptavidin-gH625 supramolecular structures was assembled for O/W NEs decoration. To this end, we first functionalized the gH625 sequence with a biotinylated poly(ethylene glycol) (PEG) chain in order to inhibit the nanocarriers clearance by eluding the mononuclear phagocyte system (MPS), as highly reported among PEG properties.22 Then, we stabilized O/W NEs with a thin layer of biotinylated poly-L-lysine and, after the addition of streptavidin, the biotin-PEG-gH625 molecule was deposited by an additive easy approach that exploits biotin-streptavidin physical interaction. Isothermal Titration Calorimetry (ITC) was used to evaluate the right amount of each component to be added following a step by step procedure. Afterwards, these nanocarriers were characterized in
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terms of their chemical and morphological features. Then, a biological study was performed in order to assess, the effectiveness of the alternative penetration pathway of the proposed O/W NEs based on enhanced cytosolic localization. To this aim, we compared the performance of O/W NE functionalized with gH625 to that of the same system conjugated with a non-active peptide in a monolayer of bEnd.3 cells. Subsequently, we confirmed the relevance of this result by loading both the systems with curcumin and comparing their anti-inflammatory activity in primary isolated cells, namely human umbilical vein endothelial cells (HUVEC). This test demonstrated that not only the anticancer activity of curcumin is higher when this molecule is delivered directly into the cytosol23 but also its anti-inflammatory effect is enhanced. Finally, in order to explore the ability of our O/W NEs to properly interact with cells in a 3D setting, we performed a sprouting angiogenesis assay based on the use of HUVEC spheroids.
RESULTS AND DISCUSSION Nanocapsule Preparation and Characterization O/W NEs and poly-L-lysine based secondary nanoemulsion (SNE) systems here used were previously reported and well characterized.13, 24 To fabricate our systems we used biotinylated polyL-lysine (PLL-biotin), then the streptavidin is added and used as a linker between the PLL-biotin and the biotinylated PEG-peptide. In Figure 1 it is shown a schematic illustration of the decorated NCs assembly. The streptavidin bridges PLL-biotin and biotin-PEG-gH625 exploiting the streptavidin-biotin affinity. PEG is added to avoid NCs clearance by mononuclear phagocytic system in order to enhance their persistence in the blood.25, 26 Moreover, the terminal peptide gH625 is able to cross the membrane bilayer, thus allowing the transport of the NCs into the cytoplasm.
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Figure 1 Schematic representation of gH625-NCs multistep assembly. In the first step, the nanoemulsion is coated with PLL-Biotin, and then in the second step, streptavidin is conjugated to the PLL-biotin based SNEs. In the third step, the previously synthesized block of biotinylated PEG-peptide (active 3a and scramble 3b) is added to the preformed NCs acting streptavidin as a linker between PLL-biotin based SNEs and the biotinylated PEG-peptide.
The functionalization of poly-L-lysine with biotin was detected by nuclear magnetic resonance (NMR) (Figure 2A). The peak at 3.0 ppm was assigned to the epsilon protons of free Lys (as confirmed by the spectrum of free poly-L-lysine shown in Figure 2B), whereas the peak at 2.9 ppm was assigned to the epsilon protons of Lys bound to biotin. The reaction scheme is reported in Figure 2C. The integration of those two peaks gives the percentage of biotin bound to poly-L-lysine in the PLL-biotin samples, showing a yield of functionalization of 5.0 %. This degree of functionalization did not affect significantly the solubility of the modified polymer and at the same time guaranteed a PEG-peptide % lower than 10 as needed to let explicate PEG anti-opsonic properties.26
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Figure 2 1H1D NMR spectra of biotin-PLL (A) and PLL (B) in 90/10 H2O/D2O. Schematic representation of poly-L-lysine functionalization with biotin (C).
The biotinylated SNE (1 wt% in oil and 0.0125 wt% in PLL-biotin) was prepared and characterized by dynamic light scattering analysis which showed good results in terms of size, polydispersity index and ζ-potential. In fact, we observed an increased size (154.3 nm) of the biotinylated SNEs as compared to the NE (137.3 nm) (Figure 3A) in combination with an inversion of charge, as expected. Indeed, the ζ-potential value resulted to be negative in the NE due to the presence of charged carboxylic acid groups along the lecithin chain (used as surfactant). Consequently, it became positive in the biotinylated SNEs due to the coating with the positively charged polymer (poly-L-lysine) (Figure 3B). In addition, DLS periodical measurements showed that the biotinylated SNE maintains its hydrodynamic diameter during the 6 weeks of investigation, thus demonstrating its good stability (Figure 3C).
Figure 3 Size data of O/W NEs (black) compared with biotinylated SNEs (red) (A). ζ-Potential data of NEs (black) compared with biotinylated SNEs (red) (B). Dimensional behaviour over time for biotinylated SNEs measured by DLS analysis (C).
In order to establish the precise amount of streptavidin able to saturate all biotin groups exposed on the biotinylated SNE surface, we carried out isothermal titration calorimetry experiments (ITC). In ACS Paragon Plus Environment
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this case, biotinylated SNE (at 0.5 wt% of oil), present in the cell, was titrated with a solution of streptavidin (10.41 µM) (Figure 4A). The ITC binding curve obtained from the integration of the heat data had a sigmoidal behaviour and was fitted with an independent and equivalent-sites model. The best-fit parameters are reported in Table 1. As shown in Table 1, the affinity between biotinylated SNE and streptavidin has very high Kd (Kd=1⁄Kb) with values in the order of nM, as expected for a biotin-streptavidin interaction.27 The thermodynamic signature, traced by the thermodynamic parameters collected in Table 1 and depicted in Figure 4B, clearly shows that the interaction is exothermic and enthalpically driven. The latter condition is counterbalanced by an unfavourable entropic contribution to Gibbs energy. The favourable enthalpy is ascribable to the formation of new interactions between biotin and streptavidin upon binding. The unfavourable entropy contribution upon binding, suggests that the critical conformational rearrangements due to the loss of conformational degrees of freedom for both the interacting molecules, is predominant. To confirm that the obtained binding curve is only due to the interaction between biotin and streptavidin, the same experiment was performed without biotin. In particular, the same SNE was titrated with a solution of streptavidin (10.41 µM). Raw data (Figure 4C) showed only small peaks of dilution demonstrating that streptavidin and SNE did not interact.
Molar enthalpy
Entropic contribution
Gibbs energy
Binding constant
∆H° (kJ mol-1)
T∆S° (kJ mol-1)
∆G° (25 °C) (kJ mol-1)
Kb (M-1)
-510.1
-463.24
-46.86
1.6*108
Table 1 Thermodynamic parameters of interaction between streptavidin and biotinylated SNE at 25 °C.
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Heat rate (µJ/s)
0,8
A
0,6 0,4 0,2
µ J*mol-1 of injected Streptavidin
0,0 0
1000
2000
3000
4000
5000
Time (s) 100
B
0 -100 -200 -300 -400 -500 0
2
4
6
8 10 12 14 16 18 20 22 24 26 Injection
0,8
Heat rate (µ J/s)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
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C
0,6 0,4 0,2 0,0 0
1000
2000
3000 Time (s)
4000
5000
Figure 4 ITC data for titration by stepwise injections of streptavidin into biotinylated SNE (0.5 wt% of oil) at 25 °C (A). Normalized heat of interaction between biotinylated SNE and streptavidin. The solid squares are the experimental data obtained through the integration of the raw data and subtracting the heat of ligand dilution into the buffer. The lines represent the best fit obtained with the independent-sites model (B). ITC data for titration by stepwise injections of streptavidin into non biotinylated SNE (0.5 wt% of oil) at 25 °C (C).
According to the NMR data, biotinylated SNE (0.5 wt% of oil and 0.00625 wt%) is 14.5 µM concentrated in biotin (corresponding to 5 % of the total amount of NH2 of poly-L-lysine). In addition, ITC data show that the same SNE saturated with streptavidin is 1.45 µM concentrated. This means that only 10% of biotin molecules are exposed on the SNE surface and able to bind the streptavidin added to the solution. ITC experiments allowed to calculate the exact amount of biotin exposed on the NC surface with respect to the total quantity. These data were of basic importance to move forward the decoration of the NCs by molecular assembly in order to avoid nonspecific interactions due to the addition of an amount of biomolecules that exceeds the available sites. ACS Paragon Plus Environment
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Regarding the peptide synthesis three glycines and a cysteine (as Fmoc-Cys(Mmt)-OH) were added to the C-terminal (gH625-GGGC). The three glycines acted as spacers in order to retain the innate peptide conformation when conjugated to the particle surface. FITC was added at the N-terminal spaced with a β-alanine in order to label the peptide sequence and to detect its conjugation with the NCs (FITC-gH625-GGGC). The PEGylated peptide was produced by selectively removing the Mmt protector group from the thiol group of the last cysteine residue, when the peptide was still linked to the resin. Then, the maleimide group of a bifunctionalized polyethylenglycol (Mal-PEGbiotin, 2000 Da) was coupled obtaining the biotin-PEGylated peptide (FITC-gH625-PEG-biotin). PEG 2000 was chosen for its well-known capability to extend the circulation time of the NCs and to decrease its accumulation into the liver.28 In literature it is reported that PEG2000-nanoemulsion had a 7-fold increased half-life after injection in rats.29 The schematic representation of solid phase peptide synthesis and the characterization by high pressure liquid chromatography are reported in the supplementary material (Figure S1). The secondary structure of gH625-GGGC and gH625GGGC-PEG was determined in water solution by CD spectroscopy in the far-UV spectral region (195–260 nm). CD spectra of gH625-GGGC revealed the presence of a random coil conformation. This spectral characteristic is compliant with the CD spectra of gH625 reported in literature30 demonstrating that the insertion of amino acids at the C-terminal does not alter peptide conformation. Further, the gH625-GGGC-PEG CD spectra showed the same trend proving that the long PEG chain does not interfere with the peptide secondary structures (Figure 5).
5 4 [θ ]*10-5 (mdeg dmol-1 cm2)*10
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0 -5 -10 -15 gH625-GGGC gH625-PEG
-20 -25 -30 -35
200
210
220
230
240
250
260
270
λ(nm)
Figure 5 CD spectra of gH625-GGGC (black) and gH625-GGGC-PEG (red) in water. The spectra were recorded using 0.2 cm path length cells (C).
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Pure FITC-gH625-PEG-biotin was then added to the streptavidin-biotinylated-SNE under sonication at a 1:1 streptavidin-biotin molar ratio. The completely functionalized NCs were finally characterized by DLS (Figure 6). Their hydrodynamic diameter was ∼156 nm, as illustrated in Table 2. In agreement with the formation of the coating, fluorescent NCs were detected by confocal microscopy (Figure S2).
Figure 6 Size data of streptavidin-biotinylated-SNEs (black) compared with gH625-NCs (red)
In the same way, pure FITC-CTRLgH625acting as negative control peptide was synthetized on solid phase, PEGylated and conjugated to the streptavidin-biotinylated-SNE surface with the same procedure used for gH625. All properties, in terms of size, PDI and ζ-potential, of all the nanocarriers are reported in Table 2.
Size (nm)
PDI
ζ-potential (mV)
O/W NE
137.3±0.56
0.041±0.003
-43.3±1.90
Biotinylated SNE
153.5±0.88
0.054±0.013
45.9±0.91
Streptavidin-biotinylated SNE
153.8±0.56
0.075±0.0063
42.9±0.83
gH625-NCs
155.9±2.00
0.099±0.01
47.5±0.60
CTRLgH625-NCs
154.8±2.90
0.068±0.02
48.6±3.39
Table 2 Size and ζ-potential measurements of NCs during the four steps of assembly.
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In Vitro Experiments To study the effect of the conjugation of the gH625 to the NC surface, we performed uptake experiments and the results were compared with those obtained using CTRLgH625-NCs. bEnd.3 cells are typically used as a blood brain barrier (BBB) in vitro 2D model due to their rapid growth end capability to form a confluent monolayer that mimics the endothelium.20, 31 In order to assess the ability of gH625 to affect the mechanisms of O/W SNE internalization, we performed confocal microscope analyses to investigate the co-localization of NCs with lysosomes. Lysosomes were stained with lysotraker (red) while NCs were stained with the FITC-conjugated peptide (green). After 24 h of incubation it was shown that gH625-NCs penetrated into the cells and were localized outside the lysosomes (Figure 7A), whereas CTRLgH625-NCs were mainly colocalized with them (Figure 7B). These data clearly indicate that the peptide changed the mechanism of nanoemulsion uptake by inducing an alternative penetration pathway as previously demonstrated with solid polystyrene nanoparticles.20 Since these experiments were performed on NCs following the emission of the FITC-peptide, in order to confirm that the entire nanocarrier was able to penetrate into the cells we performed further uptake experiments labelling also the oil core with a red lipophilic dye (Nile Red). As shown in Figure 7C-D, the fluorescence of the FITCconjugated peptide and the fluorescence of the oil containing Nile Red matched indicating that the peptide remains bound to the SNE during the uptake. Therefore, we are showing that soft matter such as O/W SNEs can be internalized by bEnd.3 cells following the extra-lysosomal path if they are conjugated with the gH625 peptide This peculiar feature may be critical to deliver high amounts of lipophilic drugs into the cells. This means that all drugs that would be potentially damaged by lysosomal environment may be delivered following the proposed approach.
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Figure 7 Colocalization of gH625-NCs (A) and CTRLgH625-NCs (B) with lysosomes in bEnd.3 cells after 24 h of incubation. Green: NPs; red: lysotracker. Uptake of gH625-NCs (C) and CTRLgH625-NCs (D) both loaded with Nile Red after 24 h incubation in bEnd.3 cells. Green: peptide; red: oil; light blue: Nuclei.
A schematic representation of the hypothesized cell uptake mechanism by means of the gH625-NCs and CTRLgH625-NCs is reported in figure 8.
Figure 8 Uptake mechanism of gH625-NCs (green spheres) and CTRLgH625-NCs (red spheres) in bEnd.3 cells.
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In order to support this hypothesis, we used a model molecule. We chose curcumin, a nutraceutical molecule, which intracellular activity is connected to its localization. In fact, it was demonstrated that curcumin exerts a higher anticancer activity when localized in the cytosol rather than into lysosomes.23 It is also known that several neurological diseases including brain tumors are related to a disruption of the blood brain barrier induced by inflammation.32 Therefore, as an applicative example, we exposed HUVEC cells to LPS-induced inflammation33. We then measured their inflammatory response to inflammation when treated with gH625-NCs or CTRLgH625-NCs. Considering the well-established anti-inflammatory activity of curcumin,34 we promoted IL-8 and IL-6 production by incubating the cells with LPS (40 ng/ml). HUVEC incubation with LPS increased significantly interleukins production through the stimulation of Toll Like Receptor type 4 (TLR4). When pretreated with free curcumin, CTRLgH625-NCs or gH625-NCs both at 5 and 15 µM concentrations, the levels of IL-6 and IL-8 decreased significantly (Figure 9). In brief, the antiinflammatory activities are summarized in the following table where it is evident always the best performance of gH625-NCs loaded with curcumin compared to CTRLgH625-NCs (Table 3).
Treatments
Reduction of interleukin secretion compared to unpretreated cells (exposed only to LPS) (%)
p value
Reduction of interleukin secretion compared to unpretreated cells (exposed only to LPS) (%)
IL-8
p value
IL-6
Free curcumin (µM) 5
9.3
0.09
15.3
0.07
15
25.2
0.004
30.3
0.007
5
11.4
0.1
18.2
0.02
15
34.3
0.03
39.2
0.002
5
40.5
0.001
42.0
0.002
15
66.5
0.0001
65.6
0.0004
CTRLgH625-NCs (µM)
gH625-NCs (µM)
Table 3 Anti-inflammatory activities for IL-6 and IL-8 for free curcumin, active and control NCs.
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As clearly showed in the table, remarkably, the most effective anti-inflammatory results were obtained with the pretreatment with gH625-NCs that reduced the production of IL-8 and IL-6 of 40,5 and 42 % at 5 µM (p=0.001; 0.002, respectively), and of 67 and 66 % at 15 µM of curcumin concentrations as compared to unpreteated cells (p=0.001; 0.004, respectively). Therefore, we carried out an uptake quantification of the two nanocarriers, by labeling the oil core with FITC, in order to elucidate the reason of the higher anti-inflammatory activity of the gH625-NCs as compared to the control ones. As clearly shown in Figure 10 (upper panel), the uptake of both fluorescent nanocarriers has a time dependent value in HUVEC cells with a linear trend over time. Interestingly, gH625-NCs showed always higher cellular uptake compared to CTRLgh625-NCS (p
